Novel formulations

ABSTRACT

There is provided inter alia an aqueous liquid pharmaceutical formulation comprising (i) an insulin compound at a concentration of 500-1000 U/ml, (ii) ionic zinc, (iii) a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C., and (iv) a non-ionic surfactant; and wherein the formulation is substantially free of EDTA and any other zinc binding species having a logK with respect to zinc ion binding of more than 12.3 at 25° C.

FIELD OF THE INVENTION

This invention relates inter alia to rapid acting aqueous liquid formulations of insulin and insulin analogues. Such compositions are suitable for the treatment of subjects suffering from diabetes mellitus, especially Type I diabetes mellitus.

BACKGROUND OF THE INVENTION

Diabetes mellitus (“diabetes”) is a metabolic disorder associated with poor control of blood sugar levels leading to hypo or hyperglycaemia. Untreated diabetes can lead to serious microvascular and macrovascular complications including coronary artery disease, peripheral artery disease, stroke, diabetic nephropathy, neuropathy and retinopathy. The two main types of diabetes are (i) Type 1 diabetes resulting from the pancreas not producing insulin for which the usual treatment is insulin replacement therapy and (ii) Type 2 diabetes where patients either produce insufficient insulin or have insulin resistance and for which treatments include insulin sensitising agents (such as metformin or pioglitazone), traditional insulin secretagogues (such as sulfonylureas), SGLT2 inhibitors (such as dapagliflozin, canagliflozin and empagliflozin) which reduce glucose absorption in the kidneys and so promote glucose excretion, GLP-1 agonists (such as exenatide and dulaglutide) which stimulate insulin release from pancreatic beta cells and DPPIV inhibitors (such as sitagliptin or vildagliptin) which inhibit breakdown of GLP-1 leading to increased insulin secretion. Patients with Type 2 diabetes may eventually require insulin replacement therapy.

For patients requiring insulin replacement therapy, a range of therapeutic options are possible. The use of recombinant human insulin has in recent times been overtaken by use of insulin analogues which have modified properties, for example, are longer acting or faster acting than normal insulin. Thus, a common regimen for a patient involves receiving a long acting basal insulin supplemented by a rapid acting insulin around mealtimes.

Insulin is a peptide hormone formed of two chains (A chain and B chain, respectively 21 and 30 amino acids in length) linked via disulfide bridges. Insulin normally exists at neutral pH in the form of a hexamer, each hexamer comprising three dimers bound together by zinc ions. Histidine residues on the insulin are known to be involved in the interaction with the zinc ions. Insulin is stored in the body in the hexameric form but the monomer form is the active form. Traditionally, therapeutic compositions of insulin have also been formulated in hexameric form in the presence of zinc ions. Typically, there are approximately three zinc cations per one insulin hexamer. It has been appreciated that the hexameric form is absorbed from the injection site considerably more slowly than the monomeric and dimeric forms. Therefore, a faster onset of insulin action can be achieved if the hexameric form is destabilised allowing a more rapid dissociation of the zinc-bound hexamer into dimers and monomers in the subcutaneous space following injection. Three insulin analogues have been genetically engineered with this principle in mind. A first is insulin lispro (Humalog®) in which residues 28 and 29 of the B chain (Pro and Lys respectively) are reversed, a second is insulin aspart (NovoRapid®) in which residue 28 of the B chain, normally Pro, is replaced by Asp, and a third is insulin glulisine (Apidra®) in which residue 3 of the B chain, normally Asn, is replaced by Lys and residue 29 of the B chain, normally Lys, is replaced by Glu.

Whilst the existing rapid acting insulin analogues can achieve a more rapid onset of action, it has been appreciated that even more rapid acting (“ultra rapid acting”) insulins can be achieved by removing the zinc cations from insulin altogether. Unfortunately, the consequence of the hexamer dissociation is typically a considerable impairment in insulin stability both with respect to physical stability (e.g. stability to aggregation) and chemical stability (e.g. stability to deamidation). For example, monomeric insulin or insulin analogues having a rapid onset of action are known to aggregate and become physically unstable very rapidly because the formulation of insoluble aggregates proceeds via monomers of insulin. Various approaches to addressing this problem have been described in the art:

U.S. Pat. No. 5,866,538 (Norup) describes insulin preparations of superior chemical stability comprising human insulin or an analogue or derivative thereof, glycerol and/or mannitol and 5 mM to 100 mM of a halogenide (e.g. NaCl).

U.S. Pat. No. 7,205,276 (Boderke) addresses the stability problems associated with preparing zinc-free formulations of insulin and insulin derivatives and analogues and describes an aqueous liquid formulation comprising at least one insulin derivative, at least one surfactant, optionally at least one preservative and optionally at least one of an isotonicizing agent, a buffer and an excipient, wherein the formulation is stable and free from or contains less than 0.4% (e.g. less than 0.2%) by weight of zinc based on the insulin content of the formulation. The preferred surfactant appears to be polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate).

US2008/0194461 (Maggio) describes formulations of peptides and polypeptides including insulin which contain an alkyl glycoside, which component is said to reduce aggregation and immunogenicity.

WO2012/006283 (Pohl) describes formulations containing insulin together with a zinc chelator such as ethylenediaminetetraacetate (EDTA). Modulating the type and quantity of EDTA is said to change the insulin absorption profile. Calcium EDTA is the preferred form of EDTA since it is said to be associated with reduced pain at the injection site and is less likely to remove calcium from the body. Preferred formulations also contain citrate which is said to further enhance absorption and to improve the chemical stability of the formulation.

US2010/0227795 (Steiner) describes a composition comprising insulin, a dissociating agent such as citric acid or sodium citrate, and a zinc chelator such as EDTA wherein the formulation has a physiological pH and is a clear aqueous solution. The formulations are said to have improved stability and rapid onset of action.

WO2015/120457 (Wilson) describes stabilized ultra-rapid acting insulin formulations comprising insulin in combination with a zinc chelator such as EDTA, a dissolution/stabilization agent such as citric acid, a magnesium salt, a zinc compound and optionally additional excipients.

Further approaches to accelerating the absorption and effect of insulin through the use of specific accelerating additives have been described:

WO91/09617 (Jorgensen) reports that nicotinamide or nicotinic acid or a salt thereof increases the speed of absorption of insulin from aqueous preparations administered parenterally.

WO2010/149772 (Olsen) describes a formulation comprising insulin, a nicotinic compound and arginine. The presence of arginine is said to improve the chemical stability of the formulation.

WO2015/171484 (Christe) describes rapid-acting formulations of insulin wherein onset of action and/or absorption of insulin is faster due to the presence of treprostinil.

US2013/0231281 (Soula) describes an aqueous solution composition comprising insulin or an insulin analogue and at least one oligosaccharide whose average degree of polymerisation is between 3 and 13 and whose polydispersity index is above 1.0, said oligosaccharide having partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable. Such a formulation is said to be rapid acting.

PCT/GB2017/051254 (Arecor Limited) describes an aqueous liquid pharmaceutical formulation comprising insulin or an insulin analogue, ionic zinc, a chelating agent and polysorbate 80.

WO2016/100042 (Eli Lilly and Company) describes a composition of human insulin or insulin analogue that includes specific concentrations of citrate, chloride, in some cases including the addition of sodium chloride, zinc and, optionally magnesium chloride and/or surfactant, said to have faster pharmacokinetic and/or pharmacodynamic action than commercial formulations of existing insulin analogue products.

Commercially available rapid-acting insulin formulations are available as 100 U/ml formulations (Humalog® (insulin lispro), NovoRapid® (also known as NovoLog®, insulin aspart) and Apidra® (insulin glulisine)) and 200 U/ml formulations (Humalog®). Formulations having a higher concentration of insulin compound are desirable e.g. for patients that require higher insulin doses, such as obese patients or patients who have developed insulin resistance. Formulations having a higher concentration of insulin are thus desirable for these categories of patients as the required high dose can be delivered in a smaller volume. Whilst the development of the 200 U/ml Humalog® formulation was an important step toward patient convenience in the situations described above, there remains a strong need to develop formulations of rapid-acting insulins at considerably higher concentrations, such as 500 U/ml or 1000 U/ml whilst maintaining the rapid onset of action.

However, a known problem associated with the use of formulations containing higher concentrations of insulin compound, in particular rapid-acting insulin compounds, is that the rapid-acting effects observed at low concentration (or low strength) formulations e.g. 100 U/ml of insulin compound, are reduced. Thus, increasing the concentration of insulin compound has been observed to lead to a slower onset of action even if the same dose is delivered, see for example de la Pefia et al. Pharmacokinetics and Pharmacodynamics of High-Dose Human Regular U-500 Insulin Versus Human Regular U-100 Insulin in Healthy Obese Subjects, Diabetes Care, 34, pp 2496-2501, 2011.

It would be desirable if analogues or formulations of insulin were available which were ultra-rapid acting, thus more closely matching the activity of physiological insulin. There also remains a need in the art to provide further, and preferably improved, formulations of insulin and insulin analogues which are rapid acting and stable. Furthermore, there is a need to provide formulations of higher concentration of insulin compound, wherein the speed of onset of action of the insulin compound is maintained.

SUMMARY OF THE INVENTION

According to the invention there is provided an aqueous liquid pharmaceutical formulation comprising (i) an insulin compound at a concentration of 500-1000 U/ml, (ii) ionic zinc, (iii) a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C., and (iv) a non-ionic surfactant; and wherein the formulation is substantially free of EDTA and any other zinc binding species having a logK with respect to zinc ion binding of more than 12.3 at 25° C. (“the formulation of the invention”). Suitably, the formulation is of low ionic strength e.g. the ionic strength of the formulation is less than 40 mM, calculated using formula Ia as described herein.

The formulations of the invention provide insulin in a form which is rapid or ultra-rapid acting with good physical and chemical stability. The formulations have a high concentration (or “high strength”) of insulin compound i.e. 500-1000 U/ml. As noted in the background discussion above, use of EDTA to chelate zinc ions in hexameric insulin does increase the rapidity of action but at the cost of greatly reduced stability. Without being limited by theory, the present inventors have appreciated that the use of zinc together with species which bind zinc less strongly can achieve similar effects in terms of speed of action and their moderately destabilising effects can be reduced or eliminated by using a non-ionic surfactant. The present inventors have further appreciated that the presence of such a zinc binding species accelerates the onset of action of a high concentration (high strength) insulin compound formulation. Put another way, the present inventors have discovered that the addition of such a zinc binding species can mitigate the delaying effect on insulin onset of action which has been observed when the concentration of insulin compound in a formulation is increased.

Formulations of the invention may be used in the treatment of subjects suffering from diabetes mellitus, particularly Type 1 diabetes mellitus especially for administration at meal times.

As can be seen from the accompanying examples, formulations of the invention are significantly more stable than corresponding formulations without non-ionic surfactant. The formulations achieve a rapid speed of action of insulin and are more stable than prior art rapid acting insulin formulations containing EDTA. Furthermore, formulations of the invention contain high concentrations of insulin compound while maintaining a rapid onset of action.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1: A chain of human insulin

SEQ ID NO: 2: B chain of human insulin

SEQ ID NO: 3: B chain of insulin lispro

SEQ ID NO: 4: B chain of insulin aspart

SEQ ID NO: 5: B chain of insulin glulisine

FIGURES

FIG. 1. Pharmacodynamic profiles of formulations 7A-7D of Example 7 in a validated diabetic Yucatan miniature pig model.

FIG. 2. Pharmacokinetic profiles of formulations 7A, 7B and 7D of Example 7 in a validated diabetic Yucatan miniature pig model.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “insulin compound” refers to insulin and insulin analogues.

As used herein, “insulin” refers to native human insulin having an A chain and a B chain as set out in SEQ ID NOs. 1 and 2 and containing and connected by disulfide bridges as in the native molecule (Cys A6-Cys A11, Cys B7 to Cys A7 and Cys-B19-Cys A20). Insulin is suitably recombinant insulin.

“Insulin analogue” refers to an analogue of insulin which is an insulin receptor agonist and has a modified amino acid sequence, such as containing 1 or 2 amino acid changes in the sequence of the A or B chain (especially the B chain). Desirably such amino acid modifications are intended to reduce affinity of the molecule for zinc and thus increase speed of action. Thus, desirably an insulin analogue has a speed of action which is the same as or preferably greater than that of insulin. The speed of action of insulin or an insulin analogue may be determined in the Diabetic Pig Pharmacokinetic/Pharmacodynamic Model (see Examples, General Methods (c)). Exemplary insulin analogues include faster acting analogues such as insulin lispro, insulin aspart and insulin glulisine. These forms of insulin have the human insulin A chain but variant B chains—see SEQ ID NOs. 3-5. Further faster acting analogues are described in EP0214826, EP0375437 and EP0678522 the contents of which are herein incorporated by reference in their entirety. Suitably, the insulin compound is not insulin glargine. Suitably, the insulin compound is not insulin degludec. Suitably, the insulin compound is a rapid-acting insulin compound, wherein “rapid-acting” is defined as an insulin compound which has a speed of action which is greater than that of native human insulin, e.g. as measured using the Diabetic Pig Pharmacokinetic/Pharmacodynamic Model (see Examples, General Methods (c)).

In one embodiment, the insulin compound is recombinant human insulin. In another embodiment, it is insulin lispro. In another embodiment, it is insulin aspart. In another embodiment, it is insulin glulisine.

The term “aqueous liquid pharmaceutical formulation”, as used herein, refers to a formulation suitable for therapeutic use in which the aqueous component is or comprises water, preferably distilled water, deionized water, water for injection, sterile water for injection or bacteriostatic water for injection. The aqueous liquid pharmaceutical formulations of the invention are solution formulations in which all components are dissolved in water.

The concentration of insulin compound in the formulation is in the range 500-1000 U/ml e.g. 800-1000 U/ml and another exemplary formulation contains insulin compound at a concentration of 1000 U/ml (around 36 mg/ml).

In one embodiment, the concentration of insulin compound in the formulation is 500-1000 U/ml e.g. >500-1000 U/ml. In one embodiment, the concentration of insulin compound in the formulation is 600-1000 U/ml, e.g. >600-1000 U/ml. In one embodiment, the concentration of insulin compound in the formulation is 700-1000 U/ml e.g. >700-1000 U/ml. In one embodiment, the concentration of insulin compound in the formulation is 750-1000 U/ml e.g. >750-1000 U/ml. In one embodiment, the concentration of insulin compound in the formulation is 800-1000 U/ml e.g. >800-1000 U/ml. In one embodiment, the concentration of insulin compound in the formulation is 900-1000 U/ml e.g. >900-1000 U/ml.

“U/ml” as used herein describes the concentration of insulin compound in terms of a unit per volume, wherein “U” is the international unit of insulin activity (see e.g. European Pharmacopoeia 5.0, Human Insulin, pp 1800-1802).

The formulations of the invention contain ionic zinc i.e. Zn2+ ions. The source of the ionic zinc will typically be a water-soluble zinc salt such as ZnCl2, ZnO, ZnSO4, Zn(NO3)2 or Zn(acetate)2 and most suitably ZnCl2 or ZnO.

The concentration of the ionic zinc in the formulation will typically be more than 0.05% e.g. more than 0.1% e.g. more than 0.2%, more than 0.3% or more than 0.4% by weight of zinc based on the weight of insulin compound in the formulation. Thus, the concentration of the ionic zinc in the formulation may be more than 0.5% by weight of zinc based on the weight of insulin compound in the formulation, for example 0.5-1%, e.g. 0.5-0.75%, e.g. 0.5-0.6% by weight of zinc based on the weight of insulin compound in the formulation. For the purpose of the calculation the weight of the counter ion to zinc is excluded.

In a formulation e.g. containing 1000 U/ml of insulin compound the concentration of the ionic zinc will typically be more than 0.15 mM e.g. more than 0.3 mM e.g. more than 0.6 mM, more than 0.9 mM or more than 1.2 mM. Thus, the concentration of the ionic zinc in the formulation may be more than 1.5 mM, for example 1.5-6.0 mM, e.g. 2.0-4.5 mM, e.g. 2.5-3.5 mM.

The formulations of the invention contain a zinc binding species. Zinc binding species should be capable of complexing ionic zinc and will have a logK metal binding stability constant with respect to zinc ion binding of 4.5-12.3 as determined at 25° C. Metal binding stability constants listed in the National Institute of Standards and Technology reference database 46 (Critically Selected Stability Constants of Metal Complexes) can be used. The database typically lists logK constants determined at 25° C. Therefore, the suitability of a zinc binding species for the present invention can be determined based on its logK metal binding stability constant with respect to zinc binding, as measured at 25° C. and as quoted by the database. The zinc binding species may also be described as an “accelerator” in the formulations according to the invention. Exemplary zinc binding species include polydendate organic anions. Thus, a preferred zinc binding species is citrate (logK=4.93) which can, for example, be employed as trisodium citrate. Further examples include pyrophosphate (logK=8.71), aspartate (logK=5.87), glutamate (logK=4.62), cysteine (logK=9.11), cystine (logK=6.67) and glutathione (logK=7.98). Other possible zinc binding species include substances that can contribute a lone pair of electrons or electron density for interaction with ionic zinc such as polydendate amines including ethylenediamine (logK=5.69), diethylenetriamine (DETA, logK=8.88) and triethylenetetramine (TETA, logK=11.95); and aromatic or heteroaromatic substances that can contribute a lone pair of electrons especially those comprising an imidazole moiety such as histidine (logK=6.51). Thus, in one embodiment, the zinc binding species having a logK with respect to zinc ion binding in the range 4.5-12.3 is selected from citrate, pyrophosphate, aspartate, glutamate, cysteine, cystine, glutathione, ethylenediamine, histidine, DETA and TETA.

In an embodiment, the zinc binding species will have a logK metal binding stability constant with respect to zinc ion binding of 4.5-10 at 25° C.

The most suitable concentration of the zinc binding species will depend on the agent and its logK value and will typically be in the range 1-100 mM. The concentration of zinc binding species can be adjusted according to the particular concentration of insulin compound present in the composition, in order to provide the desired accelerating effect.

For example, the concentration of the zinc binding species in the formulation may typically be in the range 1-50 mM. Suitably the concentration of the zinc binding species in the formulation is 10-50 mM e.g. 30-50 mM e.g. 40-50 mM, more preferably around 44 mM when the zinc binding species is citrate or histidine for insulin compound 1000 U/ml formulations.

In an embodiment, the concentration of the zinc binding species is 10 mM or more. In another embodiment, the concentration of the zinc binding species is 5-50 mM, e.g. 10-50 mM, 20-50 mM, 25-50 mM, 30-50 mM or 40-50 mM.

Anionic zinc binding species may be employed as the free acid or a salt form, such as a salt form with sodium or calcium ions, especially sodium ions.

A mixture of zinc binding species may be employed, although a single zinc binding species is preferred.

Suitably the molar ratio of ionic zinc to zinc binding species in the formulation is in the range 1:1 to 1:1000 e.g. 1:1 to 1:500 e.g. 1:1 to 1:250 or 1:3 to 1:500 e.g. 1:3 to 1:175.

The following ranges are particularly of interest especially for citrate or histidine as zinc binding species: 1:10-1:500 e.g. 1:10-1:200 e.g. 1:10 to 1:100 e.g. 1:10-1:50, e.g. 1:10 to 1:30, e.g. 1:10 to 1:20 (especially for insulin compound 1000 U/ml formulation).

For example, a formulation containing 1000 U/ml of insulin compound may contain around 3 mM of ionic zinc (i.e. around 197 μg/ml of ionic zinc, i.e. around 0.54% by weight of zinc based on the weight of insulin compound in the formulation) and around 30-60 mM e.g. 40-60 mM zinc binding species (especially citrate).

In one embodiment, the ratio of insulin compound concentration (U/ml) to zinc binding species (mM) in the formulation is in the range 100:1 to 2:1 e.g. 50:1 to 2:1 e.g. 40:1 to 2:1.

The formulations of the invention are substantially free of zinc binding species which have a logK metal binding stability constant with respect to zinc binding of more than 12.3 as determined at 25° C. Specifically, the formulations of the invention are substantially free of EDTA (logK=14.5). Further examples of zinc binding species which have a logK metal binding stability constant with respect to zinc binding of more than 12.3 to be avoided include EGTA (logK=12.6). In general formulations of the invention will be substantially free of tetradentate ligands or ligands of higher denticity. In an embodiment, the formulations of the invention are also substantially free of zinc binding species which have a logK metal binding stability constant with respect to zinc ion binding of 10-12.3 as determined at 25° C. “Substantially free” means that the concentration of zinc binding species which have a logK metal binding stability constant with respect to zinc binding as specified (such as EDTA) is less than 0.1 mM, such as less than 0.05 mM, such as less than 0.04 mM or less than 0.01 mM.

Zinc ion binding species which have acid forms (e.g. citric acid) may be introduced into the aqueous formulations of the invention in the form of a salt of the acid, such as a sodium salt (e.g. trisodium citrate). Alternatively, they can be introduced in the form of the acid with subsequent adjustment of pH to the required level. The present inventors have found that in some circumstances introducing the acid form (such as citric acid) into the formulation instead of the salt form (e.g. trisodium citrate) may have advantages in terms of providing superior chemical and physical stability. Thus, in an embodiment, the source of the citrate as zinc ion binding species is citric acid.

In one embodiment is provided an aqueous liquid pharmaceutical formulation comprising (i) an insulin compound at a concentration of 500-1000 U/ml (ii) ionic zinc, (iii) citrate as a zinc binding species at a concentration of 1 mM or more, and (iv) a non-ionic surfactant; wherein the formulation is substantially free of EDTA and any other zinc binding species having a logK with respect to zinc ion binding of more than 12.3 at 25° C., and wherein the ionic strength of the formulation is less than 40 mM, said ionic strength being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which cx is molar concentration of ion x (mol L−1), zx is the         absolute value of the charge of ion x and the sum covers all         ions (n) present in the formulation except for the zinc binding         species and the insulin compound. Suitably, the citrate is         present in the formulation at a concentration of 30-50 mM e.g.         40-50 mM.

The formulations of the invention contain a non-ionic surfactant.

A suitable class of non-ionic surfactants is the alkyl glycosides, especially dodecyl maltoside. In one embodiment, the alkyl glycoside is decyl glucopyranoside. Other alkyl glycosides include dodecyl glucoside, octyl glucoside, octyl maltoside, decyl glucoside, decyl maltoside, tridecyl glucoside, tridecyl maltoside, tetradecyl glucoside, tetradecyl maltoside, hexadecyl glucoside, hexadecyl maltoside, sucrose monooctanoate, sucrose mono decanoate, sucrose monododecanoate, sucrose monotridecanoate, sucrose monotetradecanoate and sucrose monohexadecanoate.

Another suitable class of non-ionic surfactants is the polysorbates (fatty acid esters of ethoxylated sorbitan), such as polysorbate 20 or polysorbate 80. Polysorbate 20 is a mono ester formed from lauric acid and polyoxyethylene (20) sorbitan in which the number 20 indicates the number of oxyethylene groups in the molecule. Polysorbate 80 is a mono ester formed from oleic acid and polyoxyethylene (20) sorbitan in which the number 20 indicates the number of oxyethylene groups in the molecule. Polysorbate 20 is known under a range of brand names including in particular Tween 20, and also Alkest TW 20. Polysorbate 80 is known under a range of brand names including in particular Tween 80, and also Alkest TW 80. Other suitable polysorbates include polysorbate 40 and polysorbate 60. In an embodiment, the non-ionic surfactant is other than polysorbate 80. In one embodiment, the non-ionic surfactant is other than polysorbate 20.

Another suitable class of non-ionic surfactants is block copolymers of polyethylene glycol and polypropylene glycol, also known as poloxamers, especially poloxamer 188, poloxamer 407, poloxamer 171 and poloxamer 185. Poloxamers are also known under brand names Pluronics or Koliphors. For example, poloxamer 188 is marketed as Pluronic F-68.

Another suitable class of non-ionic surfactants is alkyl ethers of polyethylene glycol, especially those known under a brand name Brij, such as selected from polyethylene glycol (2) hexadecyl ether (Brij 52), polyethylene glycol (2) oleyl ether (Brij 93) and polyethylene glycol (2) dodecyl ether (Brij L4). Other suitable Brij surfactants include polyethylene glycol (4) lauryl ether (Brij 30), polyethylene glycol (10) lauryl ether (Brij 35), polyethylene glycol (20) hexadecyl ether (Brij 58) and polyethylene glycol (10) stearyl ether (Brij 78).

Another suitable class of non-ionic surfactants are alkylphenyl ethers of polyethylene glycol, especially 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, also known under a brand name Triton X-100.

Particularly suitable are non-ionic surfactants with molecular weight of less than 1000 g/mole, especially less than 600 g/mole, such as 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100) (647 g/mole), dodecyl maltoside (511 g/mole), octyl glucoside (292 g/mole), polyethylene glycol (2) dodecyl ether (Brij L4) (362 g/mole), polyethylene glycol (2) oleyl ether (Brij 93) (357 g/mole) and polyethylene glycol (2) hexadecyl ether (Brij 52) (330 g/mole).

The concentration of the non-ionic surfactant in the formulation will typically be in the range 1-1000 μg/ml, e.g. 5-500 μg/ml, e.g. 10-200 μg/ml, such as 10-100 μg/ml especially around 50 μg/ml. In one embodiment, the non-ionic surfactant is present at a concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml, 10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200 μg/ml or 50-200 μg/ml.

In another embodiment, the concentration of insulin compound is 800-1000 U/ml and the non-ionic surfactant is present at a concentration of 50-200 μg/ml. In this embodiment, suitably the non-ionic surfactant is dodecyl maltoside.

Suitably the pH of the aqueous formulations of the invention is in the range 5.5-9.0 especially 6.5-8.0 e.g. 7.0-7.8. e.g. 7.0-7.5. In order to minimise injection pain, the pH is preferably close to physiological pH (around pH 7.4). Another pH of interest is 7.6-8.0 e.g. around 7.8. An additional pH of interest is 7.2-7.8, e.g. around 7.6.

Suitably, the composition of the invention comprises a buffer (e.g. one or more buffers) in order to stabilise the pH of the formulation, which can also be selected to enhance protein stability. In one embodiment, a buffer is selected to have a pKa close to the pH of the composition; for example, histidine is suitably employed as a buffer when the pH of the composition is in the range 5.0-7.0. Such a buffer may be employed in a concentration of 0.5-20 mM e.g. 2-5 mM. If histidine is included in the formulation as a zinc binding species it will also have a buffering role at this pH. As another example, phosphate e.g. sodium phosphate is suitably employed as a buffer when the pH of the composition is in the range 6.1-8.1. Such a buffer may be employed in a concentration of 0.5-20 mM e.g. 2-5 mM e.g. 2 mM. Alternatively, in another embodiment, the formulation of the invention is further stabilised as disclosed in WO2008/084237 (herein incorporated by reference in its entirety), which describes a formulation comprising a protein and one or more additives, characterised in that the system is substantially free of a conventional buffer, i.e. a compound with an ionisable group having a pKa within 1 unit of the pH of the formulation at the intended temperature range of storage of the composition, such as 25° C. In this embodiment, the pH of the formulation is set to a value at which the formulation has maximum measurable stability with respect to pH; the one or more additives (displaced buffers) are capable of exchanging protons with the insulin compound and have pKa values at least 1 unit more or less than the pH of the formulation at the intended temperature range of storage of the formulation. The additives may have ionisable groups having pKa between 1 to 5 pH units, preferably between 1 to 3 pH units, most preferably from 1.5 to 2.5 pH units, of the pH of the aqueous formulation at the intended temperature range of storage of the composition (e.g. 25° C.). Such additives may typically be employed at a concentration of 0.5-10 mM e.g. 2-5 mM.

The aqueous formulations of the present invention cover a wide range of osmolarity, including hypotonic, isotonic and hypertonic compositions. Preferably, the formulations of the invention are substantially isotonic. Suitably the osmolarity of the formulation is selected to minimize pain according to the route of administration e.g. upon injection. Preferred formulations have an osmolarity in the range of about 200 to about 500 mOsm/L. Preferably, the osmolarity is in the range of about 250 to about 350 mOsm/L. More preferably, the osmolarity is about 300 mOsm/L.

Tonicity of the formulation may be adjusted with a tonicity modifying agent (e.g. one or more tonicity modifying agents). Tonicity modifying agents may be charged or uncharged. Examples of charged tonicity modifying agents include salts such as a combination of sodium, potassium, magnesium or calcium ions, with chloride, sulfate, carbonate, sulfite, nitrate, lactate, succinate, acetate or maleate ions (especially sodium chloride or sodium sulphate, particularly sodium chloride). The insulin compound formulations of the invention may contain a residual NaCl concentration of 2-4 mM as a result of the use of standard acidification and subsequent neutralization steps employed in preparing insulin formulations. Amino acids such as arginine, glycine or histidine may also be used for this purpose. Charged tonicity modifying agent (e.g. NaCl) may be used at a concentration of 100-300 mM, e.g. around 150 mM. In one embodiment, the formulation of the invention comprises <10 mM chloride (e.g. sodium chloride), for example <9 mM, <8 mM, <7 mM, <6 mM or <5 mM, or is substantially free of chloride (e.g. sodium chloride) i.e. no chloride is added to the formulation beyond any chloride that may be contributed as part of pH adjustment.

Examples of uncharged tonicity modifying agents include sugars, sugar alcohols and other polyols, such as trehalose, sucrose, mannitol, glycerol, 1,2-propanediol, raffinose, lactose, dextrose, sorbitol or lactitol (especially trehalose, mannitol, glycerol or 1,2-propanediol, particularly glycerol). Uncharged tonicity modifying agent is preferably used at a concentration of 200-500 mM, e.g. around 300 mM. Another range of interest is 100-500 mM. In one embodiment, the uncharged tonicity modifying agent in the formulation is at a concentration of 100-300 mM, e.g. 150-200 mM, 170-180 mM or around 174 mM. In one embodiment, the uncharged tonicity modifying agent in the formulation is glycerol at a concentration of 100-300 mM, e.g. 150-200 mM, 170-180 mM or around 174 mM.

When the insulin compound is insulin lispro, the tonicity is suitably adjusted using an uncharged tonicity modifying agent, preferably at a concentration of 200-500 mM, e.g. around 300 mM. In this embodiment, the uncharged tonicity modifying agent is suitably selected from the group consisting of trehalose, mannitol, glycerol and 1,2-propanediol (most suitably glycerol). In another embodiment, the uncharged tonicity modifying agent is used at a concentration of 100-300 mM, e.g. 150-200 mM, 170-180 mM or around 174 mM. In one embodiment, the uncharged tonicity modifying agent is glycerol at a concentration of 100-300 mM, e.g. 150-200 mM, 170-180 mM or around 174 mM.

When the insulin compound is insulin aspart, the tonicity is suitably adjusted using an uncharged tonicity modifying agent, preferably at a concentration of 200-500 mM, e.g. around 300 mM. In this embodiment, the uncharged tonicity modifying agent is suitably selected from the group consisting of trehalose, mannitol, glycerol and 1,2-propanediol (most suitably glycerol). In another embodiment, the uncharged tonicity modifying agent is used at a concentration of 100-300 mM, e.g. 150-200 mM, 170-180 mM or around 174 mM. In one embodiment, the uncharged tonicity modifying agent is glycerol at a concentration of 100-300 mM, e.g. 150-200 mM, 170-180 mM or around 174 mM.

When the insulin compound is insulin glulisine, the tonicity is suitably adjusted using an uncharged tonicity modifying agent, preferably at a concentration of 200-500 mM, e.g. around 300 mM. In this embodiment, the uncharged tonicity modifying agent is suitably selected from the group consisting of trehalose, mannitol, glycerol and 1,2-propanediol (most suitably glycerol). In another embodiment, the uncharged tonicity modifying agent is used at a concentration of 100-300 mM, e.g. 150-200 mM, 170-180 mM or around 174 mM. In one embodiment, the uncharged tonicity modifying agent is glycerol at a concentration of 100-300 mM, e.g. 150-200 mM, 170-180 mM or around 174 mM.

The ionic strength of a formulation may be calculated according to the formula

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & \; \end{matrix}$

-   -   in which cx is molar concentration of ion x (mol L−1), zx is the         absolute value of the charge of ion x and the sum covers all         ions (n) present in the composition. The contribution of the         insulin compound itself should be ignored for the purposes of         the calculation. The contribution of the zinc binding species         should be ignored for the purposes of the calculation. Suitably,         the contribution of the ionic zinc should be included for the         purposes of the calculation. For zwitterions, the absolute value         of the charge is the total charge excluding polarity, e.g. for         glycine the possible ions have absolute charge of 0, 1 or 2 and         for aspartate the possible ions have absolute charge of 0, 1, 2         or 3.

In general, the ionic strength of the formulation is suitably less than 40 mM, e.g. less than 30 mM, less than 20 mM or less than 10 mM.

In one embodiment, the insulin compound is present at a concentration 500-1000 U/ml, e.g. >500-1000 U/ml, 600-1000 U/ml, >600-1000 U/ml, 700-1000 U/ml, >700-1000 U/ml, 750-1000 U/ml, >750-1000 U/ml, 800-1000 U/ml, >800-1000 U/ml, 900-1000 U/ml, >900-1000 U/ml or 1000 U/ml, and the ionic strength taking account of ions in the formulation except for the zinc binding species and the insulin compound is less than 40 mM, e.g. less than 30 mM, e.g. less than 20 mM, e.g. less than 10 mM such as 1-10 mM. In a further embodiment, the ionic strength taking account of ions in the formulation except for the zinc binding species and the insulin compound is less than 35 mM, less than 30 mM, less than 25 mM, less than 20 mM, less than 15 mM, or less than 10 mM, or is in the range 5-<40 mM, 5-30 mM, 5-20 mM, 2-20 mM, 1-10 mM, 2-10 mM or 5-10 mM.

When the insulin compound is insulin lispro at a concentration of 500-1000 U/ml, e.g. >500-1000 U/ml, 600-1000 U/ml, >600-1000 U/ml, 700-1000 U/ml, >700-1000 U/ml, 750-1000 U/ml, >750-1000 U/ml, 800-1000 U/ml, >800-1000 U/ml, 900-1000 U/ml, >900-1000 U/ml or 1000 U/ml, the ionic strength of the formulation is suitably kept to a minimum level since higher ionic strength formulations are less stable than lower ionic strength formulations, particularly at high concentrations of insulin. Suitably the ionic strength taking account of ions in the formulation except for the zinc binding species and the insulin compound is less than 40 mM, e.g. less than 30 mM, e.g. less than 20 mM, e.g. less than 10 mM such as 1-10 mM. In particular, the ionic strength taking account of ions in the formulation except for the zinc binding species and the insulin compound is less than 35 mM, less than 30 mM, less than 25 mM, less than 20 mM, less than 15 mM, or less than 10 mM, or is in the range 5-<40 mM, 5-30 mM, 5-20 mM, 2-20 mM, 1-10 mM, 2-10 mM or 5-10 mM.

When the insulin compound is insulin aspart at a concentration of 500-1000 U/ml e.g. >500-1000 U/ml, 600-1000 U/ml, >600-1000 U/ml, 700-1000 U/ml, >700-1000 U/ml, 750-1000 U/ml, >750-1000 U/ml, 800-1000 U/ml, >800-1000 U/ml, 900-1000 U/ml, >900-1000 U/ml or 1000 U/ml), the ionic strength of the formulation is suitably kept to a minimum level since higher ionic strength formulations are less stable than lower ionic strength formulations. Suitably the ionic strength taking account of ions in the formulation except for the zinc binding species and the insulin compound is less than 40 mM, e.g. less than 30 mM, e.g. less than 20 mM, e.g. less than 10 mM. In this case, tonicity may suitably be adjusted using an uncharged tonicity modifying agent. In particular, the ionic strength taking account of ions in the formulation except for the zinc binding species and the insulin compound is less than 35 mM, less than 30 mM, less than 25 mM, less than 20 mM, less than 15 mM, or less than 10 mM, or is in the range 5-<40 mM, 5-30 mM, 5-20 mM, 2-20 mM, 1-10 mM, 2-10 mM or 5-10 mM.

When the insulin compound is insulin glulisine at a concentration of 500-1000 U/ml e.g. >500-1000 U/ml, 600-1000 U/ml, >600-1000 U/ml, 700-1000 U/ml, >700-1000 U/ml, 750-1000 U/ml, >750-1000 U/ml, 800-1000 U/ml, >800-1000 U/ml, 900-1000 U/ml, >900-1000 U/ml or 1000 U/ml), the ionic strength of the formulation is suitably kept to a minimum level since higher ionic strength formulations may be less stable than lower ionic strength formulations. Suitably the ionic strength taking account of ions in the formulation except for the zinc binding species and the insulin compound is less than 40 mM, e.g. less than 30 mM, e.g. less than 20 mM, e.g. less than 10 mM. In this case, tonicity may suitably be adjusted using an uncharged tonicity modifying agent. In particular, the ionic strength taking account of ions in the formulation except for the zinc binding species and the insulin compound is less than 35 mM, less than 30 mM, less than 25 mM, less than 20 mM, less than 15 mM, or less than 10 mM, or is in the range 5-<40 mM, 5-30 mM, 5-20 mM, 2-20 mM, 1-10 mM, 2-10 mM or 5-10 mM.

In another embodiment, the ionic strength of a formulation may be calculated according to the formula Ib:

$I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which cx is molar concentration of ion x (mol L−1), zx is the         absolute value of the charge of ion x and the sum covers all         ions (n) present in the composition, wherein the contribution of         the insulin compound, zinc binding species and ionic zinc should         be ignored for the purposes of the calculation. For zwitterions,         the absolute value of the charge is the total charge excluding         polarity, e.g. for glycine the possible ions have absolute         charge of 0, 1 or 2 and for aspartate the possible ions have         absolute charge of 0, 1, 2 or 3.

In this embodiment, the ionic strength of the formulation is suitably less than less than 30 mM, less than 20 mM or less than 10 mM.

In one embodiment, the insulin compound is present at a concentration of 500-1000 U/ml, e.g. >500-1000 U/ml, 600-1000 U/ml, >600-1000 U/ml, 700-1000 U/ml, >700-1000 U/ml, 750-1000 U/ml, >750-1000 U/ml, 800-1000 U/ml, >800-1000 U/ml, 900-1000 U/ml, >900-1000 U/ml or 1000 U/ml, and the ionic strength taking account of ions in the formulation except for the zinc binding species, the insulin compound and the ionic zinc is less than 30 mM, e.g. less than 20 mM, e.g. less than 10 mM such as 1-10 mM. In a further embodiment, the ionic strength taking account of ions in the formulation except for the zinc binding species, the insulin compound and the ionic zinc is less than 25 mM, less than 20 mM, less than 15 mM, or less than 10 mM, or is in the range 5-<30 mM, 5-30 mM, 5-20 mM, 2-20 mM, 1-10 mM, 2-10 mM or 5-10 mM.

When the insulin compound is insulin lispro a concentration of 500-1000 U/ml, e.g. >500-1000 U/ml, 600-1000 U/ml, >600-1000 U/ml, 700-1000 U/ml, >700-1000 U/ml, 750-1000 U/ml, >750-1000 U/ml, 800-1000 U/ml, >800-1000 U/ml, 900-1000 U/ml, >900-1000 U/ml or 1000 U/ml, the ionic strength of the formulation is suitably kept to a minimum level since higher ionic strength formulations are less stable than lower ionic strength formulations, particularly at high concentrations of insulin. Suitably the ionic strength taking account of ions in the formulation except for the zinc binding species, the insulin compound and the ionic zinc is less than 30 mM, e.g. less than 20 mM, e.g. less than 10 mM such as 1-10 mM. In particular, the ionic strength taking account of ions in the formulation except for the zinc binding species, the insulin compound and the ionic zinc is less than 25 mM, less than 20 mM, less than 15 mM, or less than 10 mM, or is in the range 5-<30 mM, 5-30 mM, 5-20 mM, 2-20 mM, 1-10 mM, 2-10 mM or 5-10 mM.

When the insulin compound is insulin aspart at a concentration of 500-1000 U/ml e.g. >500-1000 U/ml, 600-1000 U/ml, >600-1000 U/ml, 700-1000 U/ml, >700-1000 U/ml, 750-1000 U/ml, >750-1000 U/ml, 800-1000 U/ml, >800-1000 U/ml, 900-1000 U/ml, >900-1000 U/ml or 1000 U/ml, the ionic strength of the formulation is suitably kept to a minimum level since higher ionic strength formulations are less stable than lower ionic strength formulations. Suitably the ionic strength taking account of ions in the formulation except for the zinc binding species, the insulin compound and the ionic zinc is less than 30 mM, e.g. less than 20 mM, e.g. less than 10 mM. In particular, the ionic strength taking account of ions in the formulation except for the zinc binding species, the insulin compound and the ionic zinc is less than 25 mM, less than 20 mM, less than 15 mM, or less than 10 mM, or is in the range 5-<30 mM, 5-30 mM, 5-20 mM, 2-20 mM, 1-10 mM, 2-10 mM or 5-10 mM. The tonicity may suitably be adjusted using an uncharged tonicity modifying agent.

When the insulin compound is insulin glulisine at a concentration of 500-1000 U/ml e.g. >500-1000 U/ml, 600-1000 U/ml, >600-1000 U/ml, 700-1000 U/ml, >700-1000 U/ml, 750-1000 U/ml, >750-1000 U/ml, 800-1000 U/ml, >800-1000 U/ml, 900-1000 U/ml, >900-1000 U/ml or 1000 U/ml, the ionic strength of the formulation is suitably kept to a minimum level since higher ionic strength formulations may be less stable than lower ionic strength formulations. Suitably the ionic strength taking account of ions in the formulation except for the zinc binding species, the insulin compound and the ionic zinc is less than 30 mM, e.g. less than 20 mM, e.g. less than 10 mM. In particular, the ionic strength taking account of ions in the formulation except for the zinc binding species, the insulin compound and ionic zinc is less than 25 mM, less than 20 mM, less than 15 mM, or less than 10 mM, or is in the range 5-<30 mM, 5-30 mM, 5-20 mM, 2-20 mM, 1-10 mM, 2-10 mM or 5-10 mM.

The formulations of the invention can optionally include a preservative (e.g. one or more preservatives), preferably phenol, m-cresol, chlorocresol, benzyl alcohol, propylparaben, methylparaben, benzalkonium chloride or benzethonium chloride. In one embodiment, the formulation includes phenol or m-cresol. In one embodiment, a mixture of preservatives is employed e.g. phenol and m-cresol.

The formulations of the invention may optionally comprise nicotinamide. The presence of nicotinamide may further increase the speed of onset of action of insulin formulated in compositions of the invention. Suitably, the concentration of nicotinamide is in the range 10-150 mM, preferably in the range 20-100 mM, such as around 80 mM.

The formulations of the invention may optionally comprise nicotinic acid or a salt thereof. The presence of nicotinic acid or a salt thereof may also further increase the speed of onset of action of insulin formulated in compositions of the invention. Suitably, the concentration of nicotinic acid or a salt thereof is in the range 10-150 mM, preferably in the range 20-100 mM, such as around 80 mM. Example salts include metal salts such as sodium, potassium and magnesium salts.

Typically, one of nicotinamide and nicotinic acid (or as salt thereof) may be included in the formulation but not both.

The formulations of the invention may optionally comprise treprostinil or a salt thereof. The presence of the treprostinil may further increase the speed of onset of action of insulin formulated in compositions of the invention. Suitably, the concentration of treprostinil in the formulation is in the range of 0.1-12 μg/ml e.g. 0.1-10 μg/ml, 0.1-9 μg/ml, 0.1-8 μg/ml, 0.1-7 μg/ml, 0.1-6 μg/ml, 0.1-5 μg/ml, 0.1-4 μg/ml, 0.1-3 μg/ml, 0.1-2 μg/ml, 0.5-2 μg/ml e.g. about 1 μg/ml.

In one embodiment, the formulation does not contain a vasodilator. In a further embodiment, the formulation does not contain treprostinil, nicotinamide, nicotinic acid or a salt thereof.

Formulations of the invention may optionally include other beneficial components including stabilising agents. For example, amino acids such as arginine or proline may be included which may have stabilising properties. Thus, in one embodiment, the formulations of the invention comprise arginine.

In an embodiment of the invention the formulations are free of acids selected from glutamic acid, ascorbic acid, succinic acid, aspartic acid, maleic acid, fumaric acid, adipic acid and acetic acid and are also free from the corresponding ionic forms of these acids.

In an embodiment of the invention the formulations are free of arginine.

In an embodiment of the invention the formulations are free of protamine and protamine salts.

In an embodiment of the invention the formulations are free of magnesium ions.

The addition of magnesium ions e.g. in the form of magnesium chloride may provide a stabilising effect. Thus, in an embodiment of the invention the formulation contains magnesium ions e.g. MgCl2.

In an embodiment of the invention the formulations are free of calcium ions.

Formulations of the invention may further comprise an additional therapeutically active agent (an “active agent”), in particular an agent of use in the treatment of diabetes (i.e. in addition to the insulin compound in particular the rapid-acting insulin compound) e.g. an amylin analogue or a GLP-1 agonist. In one embodiment, the formulation further comprises an amylin analogue such as pramlintide, suitably at a concentration of 0.1-10 mg/ml e.g. 0.2-6 mg/ml. In one embodiment, the formulation further comprises a GLP-1 agonist such as liraglutide, dulaglutide, albiglutide, exenatide or lixisenatide, suitably at a concentration of 10 μg/ml to 50 mg/ml e.g. 200 μg/ml to 10 mg/ml or 1-10 mg/ml.

Suitably the formulations of the invention are sufficiently stable that the concentration of high molecular weight species remains low upon extended storage. The term “high molecular weight species” as used herein, refers to any irreversibly formed component of the protein content which has an apparent molecular weight at least about double the molecular weight of the parent insulin compound, as detected by a suitable analytical method, such as size-exclusion chromatography. That is, high molecular weight species are multimeric aggregates of the parent insulin compound. The multimeric aggregates may comprise the parent protein molecules with considerably altered conformation or they may be an assembly of the parent protein units in the native or near-native conformation. The determination of high molecular weight species can be done using methods known in the art, including size exclusion chromatography, electrophoresis, analytical ultracentrifugation, light scattering, dynamic light scattering, static light scattering and field flow fractionation.

Suitably the formulations of the invention are sufficiently stable that they remain substantially free of visible particles after storage at 30° C. for at least one, two or three months. Visible particles are suitably detected using the 2.9.20. European Pharmacopoeia Monograph (Particulate Contamination: Visible Particles). For example, a formulation is substantially free of visible particles if it has a Visual score according to Visual Assessment Method B of 1, 2 or 3, especially 1 or 2 according to the definition given in the Examples section.

Suitably the formulations of the invention are sufficiently stable that the concentration of related species remains low upon extended storage. The term “related species” as used herein, refers to any component of the protein content formed by a chemical modification of the parent insulin compound, particularly desamido or cyclic imide forms of insulin. Related species are suitably detected by RP-HPLC.

In a preferred embodiment, the formulation of the invention retains at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99% parent insulin compound (by weight of total protein) after storage at 30° C. for one, two or three months. The percentage of insulin compound (by weight of total protein) may be determined by size-exclusion chromatography or RP-HPLC.

In a preferred embodiment, the formulation of the invention comprises no more than 4% (by weight of total protein), preferably no more than 2% high molecular weight species after storage at 30° C. for one, two or three months.

In a preferred embodiment, the formulation of the invention comprises no more than 4% (by weight of total protein), preferably no more than 2%, preferably no more than 1% A-21 desamido form of the insulin compound after storage at 30° C. for one, two or three months.

In preferred embodiments, a composition of the present invention should exhibit an increase in high molecular weight species during storage which is at least 10% lower, preferably at least 25% lower, more preferably at least 50% lower, than a composition lacking the non-ionic surfactant but otherwise identical, following storage under the same conditions (e.g. 30° C.) and length of time (e.g. one, two or three months).

In preferred embodiments, a composition of the present invention should exhibit an increase in related species during storage which is at least 10% lower, preferably at least 25% lower, more preferably at least 50% lower, than a composition lacking the non-ionic surfactant but otherwise identical, following storage under the same conditions (e.g. 30° C.) and length of time (e.g. one, two or three months).

The speed of action of a formulation of the invention may be determined in the Diabetic Pig Pharmacokinetic/Pharmacodynamic Model (see Examples, General Methods (c)). In preferred embodiments, a composition of the present invention exhibits a Tmax (i.e. time to peak insulin concentration) that is at least 20% shorter, preferably at least 30% shorter than a composition lacking the zinc binding species having a logK with respect to zinc ion binding in the range 4.5-12.3 (e.g. in the range 4.5-10) at 25° C. but otherwise identical, using the model. In preferred embodiments, a composition of the present invention exhibits an area under the curve on the pharmacodynamics profile within the first 45 minutes after injection that is at least 20% greater, preferably at least 30% greater than a composition lacking the zinc binding species having a logK with respect to zinc ion binding in the range 4.5-12.3 (e.g. in the range 4.5-10) at 25° C. but otherwise identical, using the model.

In one embodiment, the present invention provides a composition comprising (i) insulin lispro at a concentration of 500-1000 U/ml, (ii) ionic zinc, (iii) a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, and (iv) a non-ionic surfactant e.g. an alkyl glycoside; and wherein the formulation is substantially free of EDTA and any other zinc binding species having a logK with respect to zinc ion binding of more than 12.3 at 25° C., which exhibits a Tmax (i.e. time to peak insulin concentration) that is at least 20% shorter, preferably at least 30% shorter than an aqueous formulation consisting of: insulin lispro (100 U/ml), sodium phosphate (13.2 mM), glycerol (174 mM), m-cresol (29 mM), ionic zinc (19.7 μg/ml, excluding counter-ion) adjusted to pH 7.3, using the Diabetic Pig Pharmacokinetic/Pharmacodynamic Model (see Examples, General Methods (c)). In another embodiment, the present invention provides a composition comprising (i) insulin lispro at a concentration of 500-1000 U/ml, (ii) ionic zinc, (iii) a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, and (iv) a non-ionic surfactant e.g. an alkyl glycoside; and wherein the formulation is substantially free of EDTA and any other zinc binding species having a logK with respect to zinc ion binding of more than 12.3 at 25° C., which exhibits an area under the curve on the pharmacodynamics profile within the first 45 minutes after injection that is at least 20% greater, preferably at least 30% greater than an aqueous formulation consisting of: insulin lispro (100 U/ml), sodium phosphate (13.2 mM), glycerol (174 mM), m-cresol (29 mM), ionic zinc (19.7 μg/ml, excluding counter-ion) adjusted to pH 7.3, using the Diabetic Pig Pharmacokinetic/Pharmacodynamic Model (see Examples, General Methods (c)).

In one embodiment, the present invention provides a composition comprising (i) insulin aspart at a concentration of 500-1000 U/ml, (ii) ionic zinc, (iii) a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, and (iv) a non-ionic surfactant e.g. an alkyl glycoside; and wherein the formulation is substantially free of EDTA and any other zinc binding species having a logK with respect to zinc ion binding of more than 12.3 at 25° C., which exhibits a Tmax (i.e. time to peak insulin concentration) that is at least 20% shorter, preferably at least 30% shorter than an aqueous formulation consisting of: insulin aspart (100 U/ml), sodium phosphate (7 mM), glycerol (174 mM), sodium chloride (10 mM), phenol (15.9 mM), m-cresol (15.9 mM) and ionic zinc (19.7 μg/ml, excluding counter-anion) adjusted to pH 7.4, using the Diabetic Pig Pharmacokinetic/Pharmacodynamic Model (see Examples, General Methods (c)). In another embodiment, the present invention provides a composition comprising (i) insulin aspart at a concentration of 500-1000 U/ml, (ii) ionic zinc, (iii) a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, and (iv) a non-ionic surfactant e.g. an alkyl glycoside; and wherein the formulation is substantially free of EDTA and any other zinc binding species having a logK with respect to zinc ion binding of more than 12.3 at 25° C., which exhibits an area under the curve on the pharmacodynamics profile within the first 45 minutes after injection that is at least 20% greater, preferably at least 30% greater than an aqueous formulation consisting of: insulin aspart (100 U/ml), sodium phosphate (7 mM), glycerol (174 mM), sodium chloride (10 mM), phenol (15.9 mM), m-cresol (15.9 mM) and ionic zinc (19.7 μg/ml, excluding counter-anion) adjusted to pH 7.4, using the Diabetic Pig Pharmacokinetic/Pharmacodynamic Model (see Examples, General Methods (c)).

In preferred embodiments, a composition of the present invention is bioequivalent to a corresponding formulation comprising the insulin compound at 100 U/ml.

As used herein, “bioequivalent” means that the formulation of the invention has an equivalent or similar pharmacokinetic/pharmacodynamic (PK/PD) profile to a corresponding formulation. For example, the formulation of the invention exhibits a TMAX or T½MAX (measured in accordance with the Diabetic Pig Pharmacokinetic/Pharmacodynamic Model described in section (c) of General Methods) which is substantially the same as (e.g. within ±20%, e.g. within ±10%) of that of the corresponding formulation. Bioequivalence can also be established by applying the Student's t-test to the pharmacokinetic/pharmacodynamics results achieved using two different compositions as described in the diabetic pig pharmacokinetic/pharmacodynamic model described in section (c) of General Methods.

By “corresponding formulation” is meant a standard formulation i.e. a commercially available formulation of the same insulin compound at a concentration of 100 U/ml such as Humalog® (for insulin lispro) or NovoRapid® (for insulin aspart) or Apidra® (for insulin glulisine).

In one embodiment, a composition of the present invention wherein the insulin compound is insulin lispro is bioequivalent to a commercial formulation of insulin lispro at a concentration of 100 U/ml e.g. an aqueous formulation consisting of: insulin lispro (100 U/ml), sodium phosphate (13.2 mM), glycerol (174 mM), m-cresol (29 mM), ionic zinc (19.7 μg/ml, excluding counter-ion) adjusted to pH 7.3 (i.e. the formulation of Humalog®).

In one embodiment, a composition of the present invention wherein the insulin compound is insulin aspart is bioequivalent to a commercial formulation of insulin aspart at a concentration of 100 U/ml e.g. an aqueous formulation consisting of: insulin aspart (100 U/ml), sodium phosphate (7 mM), glycerol (174 mM), sodium chloride (10 mM), phenol (15.9 mM), m-cresol (15.9 mM) and ionic zinc (197 μg/ml, excluding counter-anion) adjusted to pH 7.4 (i.e. the formulation of NovoRapid®).

According to further aspects of the invention, there is provided a formulation of the invention for use in the treatment of a subject suffering from diabetes mellitus. There is also provided a method of treatment of diabetes mellitus which comprises administering to a subject in need thereof an effective amount of a formulation of the invention.

A typical insulin dose of the composition of the invention is 2-30 U, e.g. 5-15 U. Administration should suitably occur in the window between 15 minutes before eating (i.e. before start of a meal) and 15 minutes after eating (i.e. after end of a meal).

In one embodiment, the formulation of the invention is co-administered with a long acting insulin such as insulin glargine or insulin degludec, suitably at a concentration of 50-1000 U/ml e.g. 100-500 U/ml or 100-200 U/ml.

In one embodiment, the composition of the invention is for administration by intravenous injection or infusion, or subcutaneous or intramuscular injection. In one embodiment, the composition of the invention is not for administration by intranasal delivery.

An aspect of the invention is a container e.g. made of plastics or glass containing one dose or a plurality of doses of the formulation of the invention. The container can, for example, be a cartridge designed to be a replaceable item for use with an injection device.

The formulations of the invention may suitably be packaged for injection, especially sub-cutaneous or intramuscular injection. Sub-cutaneous injection is preferred. Injection may be by conventional syringe or more preferably via a pen device adapted for use by diabetic subjects. Exemplary pen devices include the Kwikpen® device and the Flexpen® device.

An aspect of the invention is an injection device, particularly a device adapted for subcutaneous or intramuscular injection, for single or multiple use comprising a container containing one dose or a plurality of doses of the formulation of the invention together with an injection needle. In an embodiment, the container is a replaceable cartridge which contains a plurality of doses. In an embodiment, the needle is replaceable e.g. after each occasion of use.

Another aspect of the invention is a medical device comprising a reservoir comprising a plurality of doses of the formulation of the invention and a pump adapted for automatic or remote operation such that upon automatic or remote operation one or more doses of the formulation of the invention is administered to the body e.g. subcutaneously or intramuscularly. Such devices may be worn on the outside of the body or implanted in the body.

Formulations of the invention may be prepared by mixing the ingredients. For example, the insulin compound may be dissolved in an aqueous formulation comprising the other components. Alternatively, the insulin compound may be dissolved in a strong acid (typically HCl), after dissolution diluted with an aqueous formulation comprising the other components, and then pH adjusted to the desired pH with addition of alkali (e.g. NaOH). As a variation on this method, a step of neutralising the acid solution may be performed before the dilution step and it may then not be necessary to adjust the pH after the dilution step (or a small adjustment only may be necessary).

According to another aspect of the invention there is provided a dry solid pharmaceutical composition suitable for reconstitution with an aqueous medium which comprises, following reconstitution, (i) an insulin compound at a concentration of 500-1000 U/ml, (ii) ionic zinc e.g. at a concentration of 0.05% or more e.g. 0.5% or more by weight of zinc based on the weight of insulin compound in the formulation, (iii) a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C., and (iv) a non-ionic surfactant; and wherein the composition is substantially free of EDTA and any other zinc binding species having a logK with respect to zinc ion binding of more than 12.3 at 25° C. Such a formulation suitably has ionic strength of less than 40 mM, said ionic strength being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which cx is molar concentration of ion x (mol L−1), zx is the         absolute value of the charge of ion x and the sum covers all         ions (n) present in the formulation except for the zinc binding         species and the insulin compound. Thus, a formulation of the         invention may be prepared by dissolving such a dry solid         pharmaceutical composition in an aqueous medium e.g. water or         saline. Such a dry solid pharmaceutical composition may be         prepared by dehydrating (e.g. freeze drying) a formulation of         the invention. The invention also provides a container         containing one dose or a plurality of doses of such a dry solid         pharmaceutical composition.

In one embodiment is provided a method of accelerating the onset of action of an aqueous liquid pharmaceutical formulation comprising (i) an insulin compound at a concentration of 500-1000 U/ml, (ii) ionic zinc, and (iii) a non-ionic surfactant; which comprises adding to the formulation a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C.

In one embodiment is provided the use of a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C. to accelerate the onset of action of an aqueous liquid pharmaceutical formulation comprising (i) an insulin compound at a concentration of 500-1000 U/ml (ii) ionic zinc, and (iii) a non-ionic surfactant, wherein the formulation is substantially free of EDTA and any other zinc binding species having a logK with respect to zinc ion binding of more than 12.3 at 25° C.

Further aspects of the invention include:

-   -   An aqueous liquid pharmaceutical formulation comprising (i) an         insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM or 10-30 mM e.g. 10-20 mM) selected from species having a         logK with respect to zinc ion binding in the range 4.5-12.3 at         25° C. e.g. citrate, and (iv) a non-ionic surfactant e.g. an         alkyl glycoside such as dodecyl maltoside; wherein the         formulation is substantially free of EDTA and any other zinc         binding species having a logK with respect to zinc ion binding         of more than 12.3 at 25° C., and wherein the ionic strength of         the formulation is less than 40 mM e.g. less than 30 mM, less         than 20 mM or less than 10 mM, said ionic strength being         calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; wherein the formulation is substantially free of         EDTA and any other zinc binding species having a logK with         respect to zinc ion binding of more than 12.3 at 25° C., and         wherein the ionic strength of the formulation is less than 40 mM         e.g. less than 30 mM, less than 20 mM or less than 10 mM, said         ionic strength being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide, and wherein the ionic strength of the formulation         is less than 40 mM e.g. less than 30 mM, less than 20 mM or less         than 10 mM, said ionic strength being calculated using the         formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation comprising (i) an         insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-10 at 25° C. e.g. citrate, and (iv)         a non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; wherein the formulation is substantially free of EDTA         and any other zinc binding species having a logK with respect to         zinc ion binding of more than 10 at 25° C., and wherein the         ionic strength of the formulation is less than 40 mM, e.g. less         than 30 mM, less than 20 mM or less than 10 mM, said ionic         strength being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-10 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; wherein the formulation is substantially free of         EDTA and any other zinc binding species having a logK with         respect to zinc ion binding of more than 10 at 25° C., and         wherein the ionic strength of the formulation is less than 40         mM, e.g. less than 30 mM, less than 20 mM or less than 10 mM,         said ionic strength being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-10 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; and wherein the ionic strength of the formulation         is less than 40 mM, e.g. less than 30 mM, less than 20 mM or         less than 10 mM, said ionic strength being calculated using the         formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation comprising (i) an         insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) citrate as a zinc binding species at a         concentration of 1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g.         30-50 mM such as 44 mM), and (iv) a non-ionic surfactant e.g. an         alkyl glycoside such as dodecyl maltoside; wherein the         formulation is substantially free of EDTA and any other zinc         binding species having a logK with respect to zinc ion binding         of more than 12.3 at 25° C., and wherein the ionic strength of         the formulation is less than 40 mM, e.g. less than 30 mM, less         than 20 mM or less than 10 mM, said ionic strength being         calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a first further embodiment, the         insulin compound is insulin lispro. In a second further         embodiment, the insulin compound is insulin aspart. In a third         further embodiment, the insulin compound is insulin glulisine.         In a fourth further embodiment, the insulin compound is         recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) citrate as a zinc binding species at a         concentration of 1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g.         30-50 mM such as 44 mM), (iv) a non-ionic surfactant e.g. an         alkyl glycoside such as dodecyl maltoside; (iv) optionally one         or more buffers e.g. phosphate such as sodium phosphate; (v)         optionally one or more preservatives e.g. phenol and         m-cresol; (vi) optionally one or more tonicity modifiers e.g.         glycerol or NaCl, and (vii) optionally one or more additional         active agents such as an amylin analogue such as pramlintide or         a GLP-1 agonist such as liraglutide, dulaglutide, albiglutide,         exenatide or lixisenatide; wherein the formulation is         substantially free of EDTA and any other zinc binding species         having a logK with respect to zinc ion binding of more than 12.3         at 25° C., and wherein the ionic strength of the formulation is         less than 40 mM, e.g. less than 30 mM, less than 20 mM or less         than 10 mM, said ionic strength being calculated using the         formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a first further embodiment, the         insulin compound is insulin lispro. In a second further         embodiment, the insulin compound is insulin aspart. In a third         further embodiment, the insulin compound is insulin glulisine.         In a fourth further embodiment, the insulin compound is         recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) citrate as a zinc binding species at a         concentration of 1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g.         30-50 mM such as 44 mM), (iv) a non-ionic surfactant e.g. an         alkyl glycoside such as dodecyl maltoside; (iv) optionally one         or more buffers e.g. phosphate such as sodium phosphate; (v)         optionally one or more preservatives e.g. phenol and         m-cresol; (vi) optionally one or more tonicity modifiers e.g.         glycerol or NaCl, and (vii) optionally one or more additional         active agents such as an amylin analogue such as pramlintide or         a GLP-1 agonist such as liraglutide, dulaglutide, albiglutide,         exenatide or lixisenatide; and wherein the ionic strength of the         formulation is less than 40 mM, e.g. less than 30 mM, less than         20 mM or less than 10 mM, said ionic strength being calculated         using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a first further embodiment, the         insulin compound is insulin lispro. In a second further         embodiment, the insulin compound is insulin aspart. In a third         further embodiment, the insulin compound is insulin glulisine.         In a fourth further embodiment, the insulin compound is         recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation comprising (i) an         insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM or 10-30 mM e.g. 10-20 mM) selected from species having a         logK with respect to zinc ion binding in the range 4.5-12.3 at         25° C. e.g. citrate, (iv) a non-ionic surfactant e.g. an alkyl         glycoside such as dodecyl maltoside; and (v) an amylin analogue         such as pramlintide; wherein the formulation is substantially         free of EDTA and any other zinc binding species having a logK         with respect to zinc ion binding of more than 12.3 at 25° C.,         and wherein the ionic strength of the formulation is less than         40 mM e.g. less than 30 mM, less than 20 mM or less than 10 mM,         said ionic strength being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         amylin analogue, e.g. pramlintide is at a concentration in the         range 0.1-10 mg/ml, e.g. 0.2-6 mg/ml. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii) an         amylin analogue such as pramlintide; wherein the formulation is         substantially free of EDTA and any other zinc binding species         having a logK with respect to zinc ion binding of more than 12.3         at 25° C., and wherein the ionic strength of the formulation is         less than 40 mM e.g. less than 30 mM, less than 20 mM or less         than 10 mM, said ionic strength being calculated using the         formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         amylin analogue, e.g. pramlintide is at a concentration in the         range 0.1-10 mg/ml, e.g. 0.2-6 mg/ml. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii) an         amylin analogue such as pramlintide; and wherein the ionic         strength of the formulation is less than 40 mM e.g. less than 30         mM, less than 20 mM or less than 10 mM, said ionic strength         being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         amylin analogue, e.g. pramlintide is at a concentration in the         range 0.1-10 mg/ml, e.g. 0.2-6 mg/ml. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation comprising (i) an         insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM or 10-30 mM e.g. 10-20 mM) selected from species having a         logK with respect to zinc ion binding in the range 4.5-12.3 at         25° C. e.g. citrate, (iv) a non-ionic surfactant e.g. an alkyl         glycoside such as dodecyl maltoside; and (v) a GLP-1 agonist         such as liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; wherein the formulation is substantially free of         EDTA and any other zinc binding species having a logK with         respect to zinc ion binding of more than 12.3 at 25° C., and         wherein the ionic strength of the formulation is less than 40 mM         e.g. less than 30 mM, less than 20 mM or less than 10 mM, said         ionic strength being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         GLP-1 agonist, e.g. liraglutide, dulaglutide, albiglutide,         exenatide or lixisenatide is at a concentration range of 10         μg/ml to 50 mg/ml, 200 μg/ml to 10 mg/ml, or 1-10 mg/ml. In one         embodiment, the surfactant is an alkyl glycoside e.g. dodecyl         maltoside, at a concentration of 10-400 μg/ml e.g. 20-400 μg/ml,         50-400 μg/ml, 10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200         μg/ml, 20-200 μg/ml or 50-200 μg/ml. In a further embodiment,         the zinc binding species is citrate or histidine (especially         citrate) and the citrate or histidine concentration in the         formulation is 10-50 mM, e.g. 30-50 mM, e.g. 40-50, e.g. around         44 mM. In a first further embodiment, the insulin compound is         insulin lispro. In a second further embodiment, the insulin         compound is insulin aspart. In a third further embodiment, the         insulin compound is insulin glulisine. In a fourth further         embodiment, the insulin compound is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii) a GLP-1         agonist such as liraglutide, dulaglutide, albiglutide, exenatide         or lixisenatide; wherein the formulation is substantially free         of EDTA and any other zinc binding species having a logK with         respect to zinc ion binding of more than 12.3 at 25° C., and         wherein the ionic strength of the formulation is less than 40 mM         e.g. less than 30 mM, less than 20 mM or less than 10 mM, said         ionic strength being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         GLP-1 agonist, e.g. liraglutide, dulaglutide, albiglutide,         exenatide or lixisenatide is at a concentration range of 10         μg/ml to 50 mg/ml, 200 μg/ml to 10 mg/ml, or 1-10 mg/ml. In one         embodiment, the surfactant is an alkyl glycoside e.g. dodecyl         maltoside, at a concentration of 10-400 μg/ml e.g. 20-400 μg/ml,         50-400 μg/ml, 10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200         μg/ml, 20-200 μg/ml or 50-200 μg/ml. In a further embodiment,         the zinc binding species is citrate or histidine (especially         citrate) and the citrate or histidine concentration in the         formulation is 10-50 mM, e.g. 30-50 mM, e.g. 40-50, e.g. around         44 mM. In a first further embodiment, the insulin compound is         insulin lispro. In a second further embodiment, the insulin         compound is insulin aspart. In a third further embodiment, the         insulin compound is insulin glulisine. In a fourth further         embodiment, the insulin compound is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii) a GLP-1         agonist such as liraglutide, dulaglutide, albiglutide, exenatide         or lixisenatide; and wherein the ionic strength of the         formulation is less than 40 mM e.g. less than 30 mM, less than         20 mM or less than 10 mM, said ionic strength being calculated         using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         GLP-1 agonist, e.g. liraglutide, dulaglutide, albiglutide,         exenatide or lixisenatide is at a concentration range of 10         μg/ml to 50 mg/ml, 200 μg/ml to 10 mg/ml, or 1-10 mg/ml. In one         embodiment, the surfactant is an alkyl glycoside e.g. dodecyl         maltoside, at a concentration of 10-400 μg/ml e.g. 20-400 μg/ml,         50-400 μg/ml, 10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200         μg/ml, 20-200 μg/ml or 50-200 μg/ml. In a further embodiment,         the zinc binding species is citrate or histidine (especially         citrate) and the citrate or histidine concentration in the         formulation is 10-50 mM, e.g. 30-50 mM, e.g. 40-50, e.g. around         44 mM. In a first further embodiment, the insulin compound is         insulin lispro. In a second further embodiment, the insulin         compound is insulin aspart. In a third further embodiment, the         insulin compound is insulin glulisine. In a fourth further         embodiment, the insulin compound is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation comprising (i) an         insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM or 10-30 mM e.g. 10-20 mM) selected from species having a         logK with respect to zinc ion binding in the range 4.5-12.3 at         25° C. e.g. citrate, (iv) a non-ionic surfactant e.g. an alkyl         glycoside such as dodecyl maltoside; and (v) treprostinil or a         salt thereof; wherein the formulation is substantially free of         EDTA and any other zinc binding species having a logK with         respect to zinc ion binding of more than 12.3 at 25° C., and         wherein the ionic strength of the formulation is less than 40 mM         e.g. less than 30 mM, less than 20 mM or less than 10 mM, said         ionic strength being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         treprostinil is at a concentration in the range 0.1-12 μg/ml         e.g. 0.1-10 μg/ml, 0.1-9 tag/ml, 0.1-8 tag/ml, 0.1-7 tag/ml,         0.1-6 tag/ml, 0.1-5 tag/ml, 0.1-4 tag/ml, 0.1-3 tag/ml, 0.1-2         μg/ml, 0.5-2 μg/ml e.g. about 1 μg/ml. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         treprostinil or a salt thereof; wherein the formulation is         substantially free of EDTA and any other zinc binding species         having a logK with respect to zinc ion binding of more than 12.3         at 25° C., and wherein the ionic strength of the formulation is         less than 40 mM e.g. less than 30 mM, less than 20 mM or less         than 10 mM, said ionic strength being calculated using the         formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         treprostinil is at a concentration in the range 0.1-12 μg/ml         e.g. 0.1-10 μg/ml, 0.1-9 μg/ml, 0.1-8 μg/ml, 0.1-7 μg/ml, 0.1-6         μg/ml, 0.1-5 μg/ml, 0.1-4 μg/ml, 0.1-3 μg/ml, 0.1-2 μg/ml, 0.5-2         μg/ml e.g. about 1 μg/ml. In one embodiment, the surfactant is         an alkyl glycoside e.g. dodecyl maltoside, at a concentration of         10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml, 10-300 μg/ml,         20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200 μg/ml or 50-200         μg/ml. In a further embodiment, the zinc binding species is         citrate or histidine (especially citrate) and the citrate or         histidine concentration in the formulation is 10-50 mM, e.g.         30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first further         embodiment, the insulin compound is insulin lispro. In a second         further embodiment, the insulin compound is insulin aspart. In a         third further embodiment, the insulin compound is insulin         glulisine. In a fourth further embodiment, the insulin compound         is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         treprostinil or a salt thereof; and wherein the ionic strength         of the formulation is less than 40 mM e.g. less than 30 mM, less         than 20 mM or less than 10 mM, said ionic strength being         calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         treprostinil is at a concentration in the range 0.1-12 μg/ml         e.g. 0.1-10 μg/ml, 0.1-9 μg/ml, 0.1-8 μg/ml, 0.1-7 μg/ml, 0.1-6         μg/ml, 0.1-5 μg/ml, 0.1-4 μg/ml, 0.1-3 μg/ml, 0.1-2 μg/ml, 0.5-2         μg/ml e.g. about 1 μg/ml. In one embodiment, the surfactant is         an alkyl glycoside e.g. dodecyl maltoside, at a concentration of         10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml, 10-300 μg/ml,         20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200 μg/ml or 50-200         μg/ml. In a further embodiment, the zinc binding species is         citrate or histidine (especially citrate) and the citrate or         histidine concentration in the formulation is 10-50 mM, e.g.         30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first further         embodiment, the insulin compound is insulin lispro. In a second         further embodiment, the insulin compound is insulin aspart. In a         third further embodiment, the insulin compound is insulin         glulisine. In a fourth further embodiment, the insulin compound         is recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation comprising (i) an         insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM or 10-30 mM e.g. 10-20 mM) selected from species having a         logK with respect to zinc ion binding in the range 4.5-12.3 at         25° C. e.g. citrate, (iv) a non-ionic surfactant e.g. an alkyl         glycoside such as dodecyl maltoside; and (v) nicotinamide,         nicotinic acid or a salt thereof; wherein the formulation is         substantially free of EDTA and any other zinc binding species         having a logK with respect to zinc ion binding of more than 12.3         at 25° C., and wherein the ionic strength of the formulation is         less than 40 mM e.g. less than 30 mM, less than 20 mM or less         than 10 mM, said ionic strength being calculated using the         formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         nicotinamide, nicotinic acid or salt thereof is at a         concentration in the range 10-150 mM, e.g. 20-100 mM such as         around 80 mM. In one embodiment, the surfactant is an alkyl         glycoside e.g. dodecyl maltoside, at a concentration of 10-400         μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml, 10-300 μg/ml, 20-300         μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200 μg/ml or 50-200 μg/ml.         In a further embodiment, the zinc binding species is citrate or         histidine (especially citrate) and the citrate or histidine         concentration in the formulation is 10-50 mM, e.g. 30-50 mM,         e.g. 40-50, e.g. around 44 mM. In a first further embodiment,         the insulin compound is insulin lispro. In a second further         embodiment, the insulin compound is insulin aspart. In a third         further embodiment, the insulin compound is insulin glulisine.         In a fourth further embodiment, the insulin compound is         recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         nicotinamide, nicotinic acid or a salt thereof; wherein the         formulation is substantially free of EDTA and any other zinc         binding species having a logK with respect to zinc ion binding         of more than 12.3 at 25° C., and wherein the ionic strength of         the formulation is less than 40 mM e.g. less than 30 mM, less         than 20 mM or less than 10 mM, said ionic strength being         calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         nicotinamide, nicotinic acid or salt thereof is at a         concentration in the range 10-150 mM, e.g. 20-100 mM such as         around 80 mM. In one embodiment, the surfactant is an alkyl         glycoside e.g. dodecyl maltoside, at a concentration of 10-400         μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml, 10-300 μg/ml, 20-300         μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200 μg/ml or 50-200 μg/ml.         In a further embodiment, the zinc binding species is citrate or         histidine (especially citrate) and the citrate or histidine         concentration in the formulation is 10-50 mM, e.g. 30-50 mM,         e.g. 40-50, e.g. around 44 mM. In a first further embodiment,         the insulin compound is insulin lispro. In a second further         embodiment, the insulin compound is insulin aspart. In a third         further embodiment, the insulin compound is insulin glulisine.         In a fourth further embodiment, the insulin compound is         recombinant human insulin.     -   An aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         nicotinamide, nicotinic acid or a salt thereof; and wherein the         ionic strength of the formulation is less than 40 mM e.g. less         than 30 mM, less than 20 mM or less than 10 mM, said ionic         strength being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         nicotinamide, nicotinic acid or salt thereof is at a         concentration in the range 10-150 mM, e.g. 20-100 mM such as         around 80 mM. In one embodiment, the surfactant is an alkyl         glycoside e.g. dodecyl maltoside, at a concentration of 10-400         μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml, 10-300 μg/ml, 20-300         μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200 μg/ml or 50-200 μg/ml.         In a further embodiment, the zinc binding species is citrate or         histidine (especially citrate) and the citrate or histidine         concentration in the formulation is 10-50 mM, e.g. 30-50 mM,         e.g. 40-50, e.g. around 44 mM. In a first further embodiment,         the insulin compound is insulin lispro. In a second further         embodiment, the insulin compound is insulin aspart. In a third         further embodiment, the insulin compound is insulin glulisine.         In a fourth further embodiment, the insulin compound is         recombinant human insulin.     -   A dry solid pharmaceutical composition suitable for         reconstitution with an aqueous medium which comprises, following         reconstitution, (i) an insulin compound at a concentration of         500-1000 U/ml e.g. 800-1000 U/ml or 1000 U/ml, (ii) ionic zinc         e.g. at a concentration of 0.05% or more e.g. 0.5% or more by         weight of zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-12.3 at 25° C. e.g. citrate,         and (iv) a non-ionic surfactant e.g. an alkyl glycoside such as         dodecyl maltoside; wherein the formulation is substantially free         of EDTA and any other zinc binding species having a logK with         respect to zinc ion binding of more than 12.3 at 25° C., and         wherein the ionic strength of the formulation is less than 40         mM, e.g. less than 30 mM, less than 20 mM or less than 10 mM,         said ionic strength being calculated using the formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   A dry solid pharmaceutical composition suitable for         reconstitution with an aqueous medium consisting of, following         reconstitution, (i) an insulin compound at a concentration of         500-1000 U/ml e.g. 800-1000 U/ml or 1000 U/ml, (ii) ionic zinc         e.g. at a concentration of 0.05% or more e.g. 0.5% or more by         weight of zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; wherein the formulation is substantially free of         EDTA and any other zinc binding species having a logK with         respect to zinc ion binding of more than 12.3 at 25° C., and         wherein the ionic strength of the formulation is less than 40         mM, e.g. less than 30 mM, less than 20 mM or less than 10 mM,         said ionic strength being calculated using the formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   A dry solid pharmaceutical composition suitable for         reconstitution with an aqueous medium consisting of, following         reconstitution, (i) an insulin compound at a concentration of         500-1000 U/ml e.g. 800-1000 U/ml or 1000 U/ml, (ii) ionic zinc         e.g. at a concentration of 0.05% or more e.g. 0.5% or more by         weight of zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-12.3 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; and wherein the ionic strength of the formulation         is less than 40 mM, e.g. less than 30 mM, less than 20 mM or         less than 10 mM, said ionic strength being calculated using the         formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   A dry solid pharmaceutical composition suitable for         reconstitution with an aqueous medium which comprises, following         reconstitution, (i) an insulin compound at a concentration of         500-1000 U/ml e.g. 800-1000 U/ml or 1000 U/ml, (ii) ionic zinc         e.g. at a concentration of 0.05% or more e.g. 0.5% or more by         weight of zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-10 at 25° C. e.g. citrate, and (iv)         a non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; wherein the formulation is substantially free of EDTA         and any other zinc binding species having a logK with respect to         zinc ion binding of more than 10 at 25° C., and wherein the         ionic strength of the formulation is less than 40 mM, e.g. less         than 30 mM, less than 20 mM or less than 10 mM, said ionic         strength being calculated using the formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   A dry solid pharmaceutical composition suitable for         reconstitution with an aqueous medium consisting of, following         reconstitution, (i) an insulin compound at a concentration of         500-1000 U/ml e.g. 800-1000 U/ml or 1000 U/ml, (ii) ionic zinc         e.g. at a concentration of 0.05% or more e.g. 0.5% or more by         weight of zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-10 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; wherein the formulation is substantially free of         EDTA and any other zinc binding species having a logK with         respect to zinc ion binding of more than 10 at 25° C., and         wherein the ionic strength of the formulation is less than 40         mM, e.g. less than 30 mM, less than 20 mM or less than 10 mM,         said ionic strength being calculated using the formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   A dry solid pharmaceutical composition suitable for         reconstitution with an aqueous medium consisting of, following         reconstitution, (i) an insulin compound at a concentration of         500-1000 U/ml e.g. 800-1000 U/ml or 1000 U/ml, (ii) ionic zinc         e.g. at a concentration of 0.05% or more e.g. 0.5% or more by         weight of zinc based on the weight of insulin compound in the         formulation, (iii) a zinc binding species at a concentration of         1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as         44 mM) selected from species having a logK with respect to zinc         ion binding in the range 4.5-10 at 25° C. e.g. citrate, (iv) a         non-ionic surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; and wherein the ionic strength of the formulation         is less than 40 mM, e.g. less than 30 mM, less than 20 mM or         less than 10 mM, said ionic strength being calculated using the         formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   A dry solid pharmaceutical composition suitable for         reconstitution with an aqueous medium which comprises, following         reconstitution, (i) an insulin compound at a concentration of         500-1000 U/ml e.g. 800-1000 U/ml or 1000 U/ml, (ii) ionic zinc         e.g. at a concentration of 0.05% or more e.g. 0.5% or more by         weight of zinc based on the weight of insulin compound in the         formulation, (iii) citrate as a zinc binding species at a         concentration of 1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g.         30-50 mM such as 44 mM), and (iv) a non-ionic surfactant e.g. an         alkyl glycoside such as dodecyl maltoside; wherein the         formulation is substantially free of EDTA and any other zinc         binding species having a logK with respect to zinc ion binding         of more than 12.3 at 25° C., and wherein the ionic strength of         the formulation is less than 40 mM, e.g. less than 30 mM, less         than 20 mM or less than 10 mM, said ionic strength being         calculated using the formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a first further embodiment, the         insulin compound is insulin lispro. In a second further         embodiment, the insulin compound is insulin aspart. In a third         further embodiment, the insulin compound is insulin glulisine.         In a fourth further embodiment, the insulin compound is         recombinant human insulin.     -   A dry solid pharmaceutical composition suitable for         reconstitution with an aqueous medium consisting of, following         reconstitution, (i) an insulin compound at a concentration of         500-1000 U/ml e.g. 800-1000 U/ml or 1000 U/ml, (ii) ionic zinc         e.g. at a concentration of 0.05% or more e.g. 0.5% or more by         weight of zinc based on the weight of insulin compound in the         formulation, (iii) citrate as a zinc binding species at a         concentration of 1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g.         30-50 mM such as 44 mM), (iv) a non-ionic surfactant e.g. an         alkyl glycoside such as dodecyl maltoside; (iv) optionally one         or more buffers e.g. phosphate such as sodium phosphate; (v)         optionally one or more preservatives e.g. phenol and         m-cresol; (vi) optionally one or more tonicity modifiers e.g.         glycerol or NaCl, and (vii) optionally one or more additional         active agents such as an amylin analogue such as pramlintide or         a GLP-1 agonist such as liraglutide, dulaglutide, albiglutide,         exenatide or lixisenatide; wherein the formulation is         substantially free of EDTA and any other zinc binding species         having a logK with respect to zinc ion binding of more than 12.3         at 25° C., and wherein the ionic strength of the formulation is         less than 40 mM, e.g. less than 30 mM, less than 20 mM or less         than 10 mM, said ionic strength being calculated using the         formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a first further embodiment, the         insulin compound is insulin lispro. In a second further         embodiment, the insulin compound is insulin aspart. In a third         further embodiment, the insulin compound is insulin glulisine.         In a fourth further embodiment, the insulin compound is         recombinant human insulin.     -   A dry solid pharmaceutical composition suitable for         reconstitution with an aqueous medium consisting of, following         reconstitution, (i) an insulin compound at a concentration of         500-1000 U/ml e.g. 800-1000 U/ml or 1000 U/ml, (ii) ionic zinc         e.g. at a concentration of 0.05% or more e.g. 0.5% or more by         weight of zinc based on the weight of insulin compound in the         formulation, (iii) citrate as a zinc binding species at a         concentration of 1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g.         30-50 mM such as 44 mM), (iv) a non-ionic surfactant e.g. an         alkyl glycoside such as dodecyl maltoside; (iv) optionally one         or more buffers e.g. phosphate such as sodium phosphate; (v)         optionally one or more preservatives e.g. phenol and         m-cresol; (vi) optionally one or more tonicity modifiers e.g.         glycerol or NaCl, and (vii) optionally one or more additional         active agents such as an amylin analogue such as pramlintide or         a GLP-1 agonist such as liraglutide, dulaglutide, albiglutide,         exenatide or lixisenatide; and wherein the ionic strength of the         formulation is less than 40 mM, e.g. less than 30 mM, less than         20 mM or less than 10 mM, said ionic strength being calculated         using the formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a first further embodiment, the         insulin compound is insulin lispro. In a second further         embodiment, the insulin compound is insulin aspart. In a third         further embodiment, the insulin compound is insulin glulisine.         In a fourth further embodiment, the insulin compound is         recombinant human insulin.     -   A method of accelerating the onset of action of an aqueous         liquid pharmaceutical formulation comprising (i) an insulin         compound at a concentration of 500-1000 U/ml e.g. 800-1000 U/ml         or 1000 U/ml, (ii) ionic zinc e.g. at a concentration of 0.05%         or more e.g. 0.5% or more by weight of zinc based on the weight         of insulin compound in the formulation and (iii) a non-ionic         surfactant e.g. an alkyl glycoside such as dodecyl maltoside;         which comprises adding a zinc binding species at a concentration         of 1 mM or more selected from species having a logK with respect         to zinc ion binding in the range 4.5-12.3 at 25° C. e.g. citrate         to the formulation suitably at a concentration of 1 mM or more         (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as 44 mM);         wherein the formulation is substantially free of EDTA and any         other zinc binding species having a logK with respect to zinc         ion binding of more than 12.3 at 25° C., wherein the ionic         strength of the formulation is less than 40 mM, e.g. less than         30 mM, less than 20 mM or less than 10 mM, said ionic strength         being calculated using the formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   A method of accelerating the onset of action of an aqueous         liquid pharmaceutical formulation consisting of (i) an insulin         compound at a concentration of 500-1000 U/ml e.g. 800-1000 U/ml         or 1000 U/ml, (ii) ionic zinc e.g. at a concentration of 0.05%         or more e.g. 0.5% or more by weight of zinc based on the weight         of insulin compound in the formulation, (iii) a non-ionic         surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; which comprises adding a zinc binding species at a         concentration of 1 mM or more selected from species having a         logK with respect to zinc ion binding in the range 4.5-12.3 at         25° C. e.g. citrate to the formulation suitably at a         concentration of 1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g.         30-50 mM such as 44 mM); wherein the formulation is         substantially free of EDTA and any other zinc binding species         having a logK with respect to zinc ion binding of more than 12.3         at 25° C., wherein the ionic strength of the formulation is less         than 40 mM, e.g. less than 30 mM, less than 20 mM or less than         10 mM, said ionic strength being calculated using the formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   A method of accelerating the onset of action of an aqueous         liquid pharmaceutical formulation consisting of (i) an insulin         compound at a concentration of 500-1000 U/ml e.g. 800-1000 U/ml         or 1000 U/ml, (ii) ionic zinc e.g. at a concentration of 0.05%         or more e.g. 0.5% or more by weight of zinc based on the weight         of insulin compound in the formulation, (iii) a non-ionic         surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; which comprises adding a zinc binding species at a         concentration of 1 mM or more selected from species having a         logK with respect to zinc ion binding in the range 4.5-12.3 at         25° C. e.g. citrate to the formulation suitably at a         concentration of 1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g.         30-50 mM such as 44 mM); wherein the ionic strength of the         formulation is less than 40 mM, e.g. less than 30 mM, less than         20 mM or less than 10 mM, said ionic strength being calculated         using the formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   A method of accelerating the onset of action of an aqueous         liquid pharmaceutical formulation comprising (i) an insulin         compound at a concentration of 500-1000 U/ml e.g. 800-1000 U/ml         or 1000 U/ml, (ii) ionic zinc e.g. at a concentration of 0.05%         or more e.g. 0.5% or more by weight of zinc based on the weight         of insulin compound in the formulation and (iii) a non-ionic         surfactant e.g. an alkyl glycoside such as dodecyl maltoside;         which comprises adding a zinc binding species at a concentration         of 1 mM or more selected from species having a logK with respect         to zinc ion binding in the range 4.5-10 at 25° C. e.g. citrate         to the formulation suitably at a concentration of 1 mM or more         (e.g. 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as 44 mM);         wherein the formulation is substantially free of EDTA and any         other zinc binding species having a logK with respect to zinc         ion binding of more than 10 at 25° C., wherein the ionic         strength of the formulation is less than 40 mM, e.g. less than         30 mM, less than 20 mM or less than 10 mM, said ionic strength         being calculated using the formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   A method of accelerating the onset of action of an aqueous         liquid pharmaceutical formulation consisting of (i) an insulin         compound at a concentration of 500-1000 U/ml e.g. 800-1000 U/ml         or 1000 U/ml, (ii) ionic zinc e.g. at a concentration of 0.05%         or more e.g. 0.5% or more by weight of zinc based on the weight         of insulin compound in the formulation, (iii) a non-ionic         surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; which comprises adding a zinc binding species at a         concentration of 1 mM or more selected from species having a         logK with respect to zinc ion binding in the range 4.5-10 at         25° C. e.g. citrate to the formulation suitably at a         concentration of 1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g.         30-50 mM such as 44 mM); wherein the formulation is         substantially free of EDTA and any other zinc binding species         having a logK with respect to zinc ion binding of more than 10         at 25° C., wherein the ionic strength of the formulation is less         than 40 mM, e.g. less than 30 mM, less than 20 mM or less than         10 mM, said ionic strength being calculated using the formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   A method of accelerating the onset of action of an aqueous         liquid pharmaceutical formulation consisting of (i) an insulin         compound at a concentration of 500-1000 U/ml e.g. 800-1000 U/ml         or 1000 U/ml, (ii) ionic zinc e.g. at a concentration of 0.05%         or more e.g. 0.5% or more by weight of zinc based on the weight         of insulin compound in the formulation, (iii) a non-ionic         surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; which comprises adding a zinc binding species at a         concentration of 1 mM or more selected from species having a         logK with respect to zinc ion binding in the range 4.5-10 at         25° C. e.g. citrate to the formulation suitably at a         concentration of 1 mM or more (e.g. 10-50 mM e.g. 20-50 mM e.g.         30-50 mM such as 44 mM); wherein the ionic strength of the         formulation is less than 40 mM, e.g. less than 30 mM, less than         20 mM or less than 10 mM, said ionic strength being calculated         using the formula:

${{Ia}\text{:}\mspace{14mu} I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   A method of accelerating the onset of action of an aqueous         liquid pharmaceutical formulation comprising (i) an insulin         compound at a concentration of 500-1000 U/ml e.g. 800-1000 U/ml         or 1000 U/ml, (ii) ionic zinc e.g. at a concentration of 0.05%         or more e.g. 0.5% or more by weight of zinc based on the weight         of insulin compound in the formulation and (iii) a non-ionic         surfactant e.g. an alkyl glycoside such as dodecyl maltoside;         which comprises adding citrate as a zinc binding species at a         concentration of 1 mM or more (suitably 10-50 mM e.g. 20-50 mM         e.g. 30-50 mM such as 44 mM); wherein the formulation is         substantially free of EDTA and any other zinc binding species         having a logK with respect to zinc ion binding of more than 12.3         at 25° C., and wherein the ionic strength of the formulation is         less than 40 mM, e.g. less than 30 mM, less than 20 mM or less         than 10 mM, said ionic strength being calculated using the         formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a first further embodiment, the         insulin compound is insulin lispro. In a second further         embodiment, the insulin compound is insulin aspart. In a third         further embodiment, the insulin compound is insulin glulisine.         In a fourth further embodiment, the insulin compound is         recombinant human insulin.     -   A method of accelerating the onset of action of an aqueous         liquid pharmaceutical formulation consisting of (i) an insulin         compound at a concentration of 500-1000 U/ml e.g. 800-1000 U/ml         or 1000 U/ml, (ii) ionic zinc e.g. at a concentration of 0.05%         or more e.g. 0.5% or more by weight of zinc based on the weight         of insulin compound in the formulation, (iii) a non-ionic         surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; which comprises adding citrate as a zinc binding         species which may preferably be incorporated into the         formulation as citric acid) at a concentration of 1 mM or more         (suitably 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as 44 mM);         wherein the formulation is substantially free of EDTA and any         other zinc binding species having a logK with respect to zinc         ion binding of more than 12.3 at 25° C., and wherein the ionic         strength of the formulation is less than 40 mM, e.g. less than         30 mM, less than 20 mM or less than 10 mM, said ionic strength         being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a first further embodiment, the         insulin compound is insulin lispro. In a second further         embodiment, the insulin compound is insulin aspart. In a third         further embodiment, the insulin compound is insulin glulisine.         In a fourth further embodiment, the insulin compound is         recombinant human insulin.     -   A method of accelerating the onset of action of an aqueous         liquid pharmaceutical formulation consisting of (i) an insulin         compound at a concentration of 500-1000 U/ml e.g. 800-1000 U/ml         or 1000 U/ml, (ii) ionic zinc e.g. at a concentration of 0.05%         or more e.g. 0.5% or more by weight of zinc based on the weight         of insulin compound in the formulation, (iii) a non-ionic         surfactant e.g. an alkyl glycoside such as dodecyl         maltoside; (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; which comprises adding citrate as a zinc binding         species which may preferably be incorporated into the         formulation as citric acid) at a concentration of 1 mM or more         (suitably 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as 44 mM);         and wherein the ionic strength of the formulation is less than         40 mM, e.g. less than 30 mM, less than 20 mM or less than 10 mM,         said ionic strength being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a first further embodiment, the         insulin compound is insulin lispro. In a second further         embodiment, the insulin compound is insulin aspart. In a third         further embodiment, the insulin compound is insulin glulisine.         In a fourth further embodiment, the insulin compound is         recombinant human insulin.     -   Use of a zinc binding species at a concentration of 1 mM or more         (suitably 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as 44 mM)         selected from species having a logK with respect to zinc ion         binding in the range 4.5-12.3 at 25° C. e.g. citrate wherein the         formulation is substantially free of EDTA and any other zinc         binding species having a logK with respect to zinc ion binding         of more than 12.3 at 25° C.; to accelerate the onset of action         of an aqueous liquid pharmaceutical formulation comprising (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the formulation         and (iii) a non-ionic surfactant e.g. an alkyl glycoside such as         dodecyl maltoside, wherein the ionic strength of the formulation         is less than 40 mM, e.g. less than 30 mM, less than 20 mM or         less than 10 mM, said ionic strength being calculated using the         formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   Use of a zinc binding species at a concentration of 1 mM or more         (suitably 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as 44 mM)         selected from species having a logK with respect to zinc ion         binding in the range 4.5-12.3 at 25° C. e.g. citrate wherein the         formulation is substantially free of EDTA and any other zinc         binding species having a logK with respect to zinc ion binding         of more than 12.3 at 25° C.; to accelerate the onset of action         of an aqueous liquid pharmaceutical formulation consisting         of (i) an insulin compound at a concentration of 500-1000 U/ml         e.g. 800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation, (iii) a non-ionic surfactant e.g. an alkyl         glycoside such as dodecyl maltoside, (iv) optionally one or more         buffers e.g. phosphate such as sodium phosphate; (v) optionally         one or more preservatives e.g. phenol and m-cresol; (vi)         optionally one or more tonicity modifiers e.g. glycerol or NaCl,         and (vii) optionally one or more additional active agents such         as an amylin analogue such as pramlintide or a GLP-1 agonist         such as liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; wherein the ionic strength of the formulation is         less than 40 mM, e.g. less than 30 mM, less than 20 mM or less         than 10 mM, said ionic strength being calculated using the         formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   Use of a zinc binding species at a concentration of 1 mM or more         (suitably 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as 44 mM)         selected from species having a logK with respect to zinc ion         binding in the range 4.5-12.3 at 25° C. e.g. citrate; to         accelerate the onset of action of an aqueous liquid         pharmaceutical formulation consisting of (i) an insulin compound         at a concentration of 500-1000 U/ml e.g. 800-1000 U/ml or 1000         U/ml, (ii) ionic zinc e.g. at a concentration of 0.05% or more         e.g. 0.5% or more by weight of zinc based on the weight of         insulin compound in the formulation, (iii) a non-ionic         surfactant e.g. an alkyl glycoside such as dodecyl         maltoside, (iv) optionally one or more buffers e.g. phosphate         such as sodium phosphate; (v) optionally one or more         preservatives e.g. phenol and m-cresol; (vi) optionally one or         more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; wherein the ionic strength of the formulation is         less than 40 mM, e.g. less than 30 mM, less than 20 mM or less         than 10 mM, said ionic strength being calculated using the         formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   Use of a zinc binding species at a concentration of 1 mM or more         (suitably 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as 44 mM)         selected from species having a logK with respect to zinc ion         binding in the range 4.5-10 at 25° C. e.g. citrate, wherein the         formulation is substantially free of EDTA and any other zinc         binding species having a logK with respect to zinc ion binding         of more than 10 at 25° C.; to accelerate the onset of action of         an aqueous liquid pharmaceutical formulation comprising (i) an         insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the formulation         and (iii) a non-ionic surfactant e.g. an alkyl glycoside such as         dodecyl maltoside, wherein the ionic strength of the formulation         is less than 40 mM, e.g. less than 30 mM, less than 20 mM or         less than 10 mM, said ionic strength being calculated using the         formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   Use of a zinc binding species at a concentration of 1 mM or more         (suitably 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as 44 mM)         selected from species having a logK with respect to zinc ion         binding in the range 4.5-10 at 25° C. e.g. citrate, wherein the         formulation is substantially free of EDTA and any other zinc         binding species having a logK with respect to zinc ion binding         of more than 10 at 25° C.; to accelerate the onset of action of         an aqueous liquid pharmaceutical formulation consisting of (i)         an insulin compound at a concentration of 500-1000 U/ml e.g.         800-1000 U/ml or 1000 U/ml, (ii) ionic zinc e.g. at a         concentration of 0.05% or more e.g. 0.5% or more by weight of         zinc based on the weight of insulin compound in the         formulation (iii) a non-ionic surfactant e.g. an alkyl glycoside         such as dodecyl maltoside, (iv) optionally one or more buffers         e.g. phosphate such as sodium phosphate; (v) optionally one or         more preservatives e.g. phenol and m-cresol; (vi) optionally one         or more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; wherein the ionic strength of the formulation is         less than 40 mM, e.g. less than 30 mM, less than 20 mM or less         than 10 mM, said ionic strength being calculated using the         formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   Use of a zinc binding species at a concentration of 1 mM or more         (suitably 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as 44 mM)         selected from species having a logK with respect to zinc ion         binding in the range 4.5-10 at 25° C. e.g. citrate; to         accelerate the onset of action of an aqueous liquid         pharmaceutical formulation consisting of (i) an insulin compound         at a concentration of 500-1000 U/ml e.g. 800-1000 U/ml or 1000         U/ml, (ii) ionic zinc e.g. at a concentration of 0.05% or more         e.g. 0.5% or more by weight of zinc based on the weight of         insulin compound in the formulation (iii) a non-ionic surfactant         e.g. an alkyl glycoside such as dodecyl maltoside, (iv)         optionally one or more buffers e.g. phosphate such as sodium         phosphate; (v) optionally one or more preservatives e.g. phenol         and m-cresol; (vi) optionally one or more tonicity modifiers         e.g. glycerol or NaCl, and (vii) optionally one or more         additional active agents such as an amylin analogue such as         pramlintide or a GLP-1 agonist such as liraglutide, dulaglutide,         albiglutide, exenatide or lixisenatide; wherein the ionic         strength of the formulation is less than 40 mM, e.g. less than         30 mM, less than 20 mM or less than 10 mM, said ionic strength         being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a further embodiment, the zinc binding         species is citrate or histidine (especially citrate) and the         citrate or histidine concentration in the formulation is 10-50         mM, e.g. 30-50 mM, e.g. 40-50, e.g. around 44 mM. In a first         further embodiment, the insulin compound is insulin lispro. In a         second further embodiment, the insulin compound is insulin         aspart. In a third further embodiment, the insulin compound is         insulin glulisine. In a fourth further embodiment, the insulin         compound is recombinant human insulin.     -   Use of a citrate as a zinc binding species at a concentration of         1 mM (suitably 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as 44         mM); wherein the formulation is substantially free of EDTA and         any other zinc binding species having a logK with respect to         zinc ion binding of more than 12.3 at 25° C. to accelerate the         onset of action of an aqueous liquid pharmaceutical formulation         comprising (i) an insulin compound at a concentration of         500-1000 U/ml e.g. 800-1000 U/ml or 1000 U/ml, (ii) ionic zinc         e.g. at a concentration of 0.05% or more e.g. 0.5% or more by         weight of zinc based on the weight of insulin compound in the         formulation and (iii) a non-ionic surfactant e.g. an alkyl         glycoside such as dodecyl maltoside, wherein the ionic strength         of the formulation is less than 40 mM, e.g. less than 30 mM,         less than 20 mM or less than 10 mM, said ionic strength being         calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound.     -   Use of a citrate as a zinc binding species at a concentration of         1 mM (suitably 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as 44         mM); wherein the formulation is substantially free of EDTA and         any other zinc binding species having a logK with respect to         zinc ion binding of more than 12.3 at 25° C. to accelerate the         onset of action of an aqueous liquid pharmaceutical formulation         consisting of (i) an insulin compound at a concentration of         500-1000 U/ml e.g. 800-1000 U/ml or 1000 U/ml, (ii) ionic zinc         e.g. at a concentration of 0.05% or more e.g. 0.5% or more by         weight of zinc based on the weight of insulin compound in the         formulation (iii) a non-ionic surfactant e.g. an alkyl glycoside         such as dodecyl maltoside, (iv) optionally one or more buffers         e.g. phosphate such as sodium phosphate; (v) optionally one or         more preservatives e.g. phenol and m-cresol; (vi) optionally one         or more tonicity modifiers e.g. glycerol or NaCl, and (vii)         optionally one or more additional active agents such as an         amylin analogue such as pramlintide or a GLP-1 agonist such as         liraglutide, dulaglutide, albiglutide, exenatide or         lixisenatide; wherein the ionic strength of the formulation is         less than 40 mM, e.g. less than 30 mM, less than 20 mM or less         than 10 mM, said ionic strength being calculated using the         formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a first further embodiment, the         insulin compound is insulin lispro. In a second further         embodiment, the insulin compound is insulin aspart. In a third         further embodiment, the insulin compound is insulin glulisine.         In a fourth further embodiment, the insulin compound is         recombinant human insulin.     -   Use of a citrate as a zinc binding species at a concentration of         1 mM (suitably 10-50 mM e.g. 20-50 mM e.g. 30-50 mM such as 44         mM); to accelerate the onset of action of an aqueous liquid         pharmaceutical formulation consisting of (i) an insulin compound         at a concentration of 500-1000 U/ml e.g. 800-1000 U/ml or 1000         U/ml, (ii) ionic zinc e.g. at a concentration of 0.05% or more         e.g. 0.5% or more by weight of zinc based on the weight of         insulin compound in the formulation (iii) a non-ionic surfactant         e.g. an alkyl glycoside such as dodecyl maltoside, (iv)         optionally one or more buffers e.g. phosphate such as sodium         phosphate; (v) optionally one or more preservatives e.g. phenol         and m-cresol; (vi) optionally one or more tonicity modifiers         e.g. glycerol or NaCl, and (vii) optionally one or more         additional active agents such as an amylin analogue such as         pramlintide or a GLP-1 agonist such as liraglutide, dulaglutide,         albiglutide, exenatide or lixisenatide; wherein the ionic         strength of the formulation is less than 40 mM, e.g. less than         30 mM, less than 20 mM or less than 10 mM, said ionic strength         being calculated using the formula:

$\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$

-   -   in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x)         is the absolute value of the charge of ion x and the sum covers         all ions (n) present in the formulation except for the zinc         binding species and the insulin compound. In one embodiment, the         surfactant is an alkyl glycoside e.g. dodecyl maltoside, at a         concentration of 10-400 μg/ml e.g. 20-400 μg/ml, 50-400 μg/ml,         10-300 μg/ml, 20-300 μg/ml, 50-300 μg/ml, 10-200 μg/ml, 20-200         μg/ml or 50-200 μg/ml. In a first further embodiment, the         insulin compound is insulin lispro. In a second further         embodiment, the insulin compound is insulin aspart. In a third         further embodiment, the insulin compound is insulin glulisine.         In a fourth further embodiment, the insulin compound is         recombinant human insulin.

Formulations of the invention in at least some embodiments are expected to have one or more of the following advantageous properties:

-   -   rapid speed of action, typically faster than normal human         insulin, upon administration to a subject;     -   rapid speed of action, typically as fast as a corresponding         formulation with insulin compound concentration of 100 U/ml;     -   high insulin concentration while maintaining a rapid speed of         action;     -   good physical stability upon storage, especially as measured by         the amount of HMWS or visual detection of particles;     -   good chemical stability upon storage, especially as measured by         the amount of related products e.g. products of deamidation.

Abbreviations

-   -   DETA diethylenetriamine     -   EDTA ethylenediaminetetraacetate     -   EGTA ethyleneglycoltetraacetate     -   HPLC high performance liquid chromatography     -   HMWS high molecular weight species     -   RP reverse phase     -   SEC size-exclusion chromatography     -   TETA triethylenetetramine     -   PD pharmacodynamic     -   PK pharmacokinetic

EXAMPLES

General Methods

(a) Size Exclusion Chromatography (SEC)

Ultra-high performance size exclusion chromatography of insulin preparations was performed using the Waters ACQUITY H-class Bio UPLC® system with a 1.7 μm Ethylene Bridged Hybrid 125 Å pore packing material in a 300 mm by 4.6 mm column. The column was equilibrated in 0.65 mg/ml L-arginine, 20% v/v acetonitrile, 15% v/v glacial acetic acid mobile phase and 10 μl of sample, acidified with 0.01M HCl, was analysed at 0.4 mL/min, with 276 nm UV detection. All analyses were performed at ambient temperature.

(b) Reversed-Phase Chromatography (RP-HPLC)

Ultra-high performance reverse phase chromatography was performed using the Waters ACQUITY H-class Bio UPLC® system with a 1.7 μm Ethylene Bridged Hybrid particle, 130 Å pore resin trifunctionally immobilised with a C18 ligand in a 50 mm by 2.1 mm column. Insulin samples were bound in a 82% w/v Na₂SO₄, 18% v/v acetonitrile, pH 2.3 mobile phase and eluted in 50% w/v Na₂SO₄, 50% v/v acetonitrile gradient flow. 2 μl of sample was acidified with 0.01M HCl and analysed at 0.61 mL/min, with 214 nm UV detection. All analyses were performed at 40° C.

(c) The Diabetic Pig Pharmacokinetic/Pharmacodynamic Model: Method for Determining Speed of Action:

10 male diabetic Yucatan miniature pigs were used. Pigs were injected subcutaneously with a sample of the test formulation and blood was taken (1 or 2 ml) at various time-points (min) with respect to the injection up to around 240 min after the injection. For pharmacodynamics profile, serum was analysed for glucose (using a commercially available glucometer). For pharmacokinetic profile, insulin concentration was determined in the serum using an immunoassay.

In order to evaluate the formulations for bioequivalence, mean values of T_(MAX) (i.e. time to reach the maximum insulin concentration in serum) and corresponding standard deviation were calculated across the whole set of 10 pigs used in the study. Similarly, mean values of T_(1/2MAX) (i.e. time to reach half of the maximum concentration) and corresponding standard deviation were calculated across the whole set of 10 pigs used in the study. Student t-test (95% confidence interval) was subsequently applied to allow assessment of bioequivalence between any two formulations tested. If the p-value of the t-test applied to the results populations of two samples was >0.05 the samples were considered bioequivalent, if the result was <0.05 then the samples were considered non-bioequivalent.

(d) Visual Assessment

Visible particles are suitably detected using the 2.9.20. European Pharmacopoeia Monograph (Particulate Contamination: Visible Particles). The apparatus required consists of a viewing station comprising:

-   -   a matt black panel of appropriate size held in a vertical         position     -   a non-glare white panel of appropriate size held in a vertical         position next to the black panel     -   an adjustable lampholder fitted with a suitable, shaded,         white-light source and with a suitable light diffuser (a viewing         illuminator containing two 13 W fluorescent tubes, each 525 mm         in length, is suitable). The intensity of illumination at the         viewing point is maintained between 2000 lux and 3750 lux.

Any adherent labels are removed from the container and the outside washed and dried. The container is gently swirled or inverted, ensuring that air bubbles are not introduced, and observed for about 5 s in front of the white panel. The procedure is repeated in front of the black panel. The presence of any particles is recorded.

The visual scores are ranked as follows:

Visual Assessment Scoring Method A

Visual score 1: clear solution free of visible particles

Visual score 2: slight particle formation

Visual score 3: more significant precipitation

Visual Assessment Scoring Method B

Visual score 1: Clear solution, virtually free of particles

Visual score 2: ˜5 very small particles

Visual score 3: ˜10-20 very small particles

Visual score 4: 20˜50 particles, including larger particles

Visual score 5: >50 particles, including larger particles

Whilst the particles in samples with visual scores 4 and 5 are clearly detectable on casual visual assessment under normal light, samples with visual score 1-3 generally appear as clear solutions on the same assessment. Samples with visual scores 1-3 are considered to be “Pass”; samples with visual score 4-5 are considered to be “Fail”.

Example 1—Example Formulations

The following example formulations may be prepared:

Example A

Insulin aspart 1000 U/ml Sodium phosphate 2 mM phenol 15.9 mM m-cresol 15.9 mM Ionic zinc (as ZnCl₂) 197 μg/ml (3 mM), equals 0.55% (w/w) based on the weight of insulin compound in the formulation Citric acid 44 mM Glycerol 174 mM Surfactant Selected from A1, A2 or A3 (see below) Water for injection qs Residual NaCl Acidification and subsequent neutralisation during preparation results in formation of 2-4 mM NaCl pH adjusted to 7.4 Example A1: surfactant = dodecyl maltoside (0.05 mg/ml) Example A2: surfactant = polysorbate 20 (Tween 20) (0.05 mg/ml) Example A3: surfactant = polyethylene glycol (2) dodecyl ether (Brij L4) (0.05 mg/ml)

Example B

Insulin aspart 1000 U/ml Sodium phosphate 2 mM phenol 15.9 mM m-cresol 15.9 mM Ionic zinc (as ZnCl₂) 197 μg/ml (3 mM), equals 0.55% (w/w) based on the weight of insulin compound in the formulation Citric acid 44 mM Glycerol 174 mM Surfactant Selected from B1, B2 or B3 (see below) Water for injection qs Residual NaCl Acidification and subsequent neutralisation during preparation results in formation of 2-4 mM NaCl pH adjusted to 7.8 Example B1: surfactant = dodecyl maltoside (0.05 mg/ml) Example B2: surfactant = polysorbate 20 (Tween 20) (0.05 mg/ml) Example B3: surfactant = polyethylene glycol (2) dodecyl ether (Brij L4) (0.05 mg/ml)

Example C

Insulin lispro 1000 U/ml Sodium phosphate 2 mM phenol 15.9 mM m-cresol 15.9 mM Ionic zinc (as ZnCl₂) 197 μg/ml (3 mM), equals 0.55% (w/w) based on the weight of insulin compound in the formulation Citric acid 44 mM Glycerol 174 mM Surfactant Selected from C1, C2 or C3 (see below) Water for injection qs Residual NaCl Acidification and subsequent neutralisation during preparation results in formation of 2-4 mM NaCl pH adjusted to 7.4 Example C1: surfactant = dodecyl maltoside (0.05 mg/ml) Example C2: surfactant = polysorbate 20 (Tween 20) (0.05 mg/ml) Example C3: surfactant = polyethylene glycol (2) dodecyl ether (Brij L4) (0.05 mg/ml)

Example D

Insulin lispro 1000 U/ml Sodium phosphate 2 mM phenol 15.9 mM m-cresol 15.9 mM Ionic zinc (as ZnCl₂) 197 μg/ml (3 mM), equals 0.55% (w/w) based on the weight of insulin compound in the formulation Citric acid 44 mM Glycerol 174 mM Surfactant Selected from D1, D2 or D3 (see below) Water for injection qs Residual NaCl Acidification and subsequent neutralisation during preparation results in formation of 2-4 mM NaCl pH adjusted to 7.8 Example D1: surfactant = dodecyl maltoside (0.05 mg/ml) Example D2: surfactant = polysorbate 20 (Tween 20) (0.05 mg/ml) Example D3: surfactant = polyethylene glycol (2) dodecyl ether (Brij L4) (0.05 mg/ml)

Insulin glulisine 1000 U/ml Sodium phosphate 2 mM phenol 15.9 mM m-cresol 15.9 mM Ionic zinc (as ZnCl₂) 197 μg/ml (3 mM), equals 0.55% (w/w) based on the weight of insulin compound in the formulation Citric acid 44 mM Glycerol 174 mM Surfactant Selected from E1, E2 or E3 (see below) Water for injection qs Residual NaCl Acidification and subsequent neutralisation during preparation results in formation of 2-4 mM NaCl pH adjusted to 7.4 Example E1: surfactant = dodecyl maltoside (0.05 mg/ml) Example E2: surfactant = polysorbate 20 (Tween 20) (0.05 mg/ml) Example E3: surfactant = polyethylene glycol (2) dodecyl ether (Brij L4) (0.05 mg/ml)

Example F

Insulin glulisine 1000 U/ml Sodium phosphate 2 mM phenol 15.9 mM m-cresol 15.9 mM Ionic zinc (as ZnCl₂) 197 μg/ml (3 mM), equals 0.55% (w/w) based on the weight of insulin compound in the formulation Citric acid 44 mM Glycerol 174 mM Surfactant Selected from F1, F2 or F3 (see below) Water for injection qs Residual NaCl Acidification and subsequent neutralisation during preparation results in formation of 2-4 mM NaCl pH adjusted to 7.8 Example F1: surfactant = dodecyl maltoside (0.05 mg/ml) Example F2: surfactant = polysorbate 20 (Tween 20) (0.05 mg/ml) Example F3: surfactant = polyethylene glycol (2) dodecyl ether (Brij L4) (0.05 mg/ml)

Example G

Insulin aspart 1000 U/ml Sodium phosphate 2 mM phenol 15.9 mM m-cresol 15.9 mM Ionic zinc (as ZnCl₂) 197 μg/ml (3 mM), equals 0.55% (w/w) based on the weight of insulin compound in the formulation TETA 5 mM Glycerol 174 mM Surfactant Selected from G1, G2 or G3 (see below) Water for injection qs Residual NaCl Acidification and subsequent neutralisation during preparation results in formation of 2-4 mM NaCl pH adjusted to 7.4 Example G1: surfactant = dodecyl maltoside (0.05 mg/ml) Example G2: surfactant = polysorbate 20 (Tween 20) (0.05 mg/ml) Example G3: surfactant = polyethylene glycol (2) dodecyl ether (Brij L4) (0.05 mg/ml)

Example H

Insulin aspart 1000 U/ml Sodium phosphate 2 mM phenol 15.9 mM m-cresol 15.9 mM Ionic zinc (as ZnCl₂) 197 μg/ml (3 mM), equals 0.55% (w/w) based on the weight of insulin compound in the formulation TETA 5 mM Glycerol 174 mM Surfactant Selected from H1, H2 or H3 (see below) Water for injection qs Residual NaCl Acidification and subsequent neutralisation during preparation results in formation of 2-4 mM NaCl pH adjusted to 7.8 Example H1: surfactant = dodecyl maltoside (0.05 mg/ml) Example H2: surfactant = polysorbate 20 (Tween 20) (0.05 mg/ml) Example H3: surfactant = polyethylene glycol (2) dodecyl ether (Brij L4) (0.05 mg/ml)

Example I

Insulin aspart 1000 U/ml Sodium phosphate 2 mM phenol 15.9 mM m-cresol 15.9 mM Ionic zinc (as ZnCl₂) 197 μg/ml (3 mM), equals 0.55% (w/w) based on the weight of insulin compound in the formulation DETA 5 mM Glycerol 174 mM Surfactant Selected from I1, I2 or I3 (see below) Water for injection qs Residual NaCl Acidification and subsequent neutralisation during preparation results in formation of 2-4 mM NaCl pH adjusted to 7.4 Example I1: surfactant = dodecyl malto side (0.05 mg/ml) Example I2: surfactant = polysorbate 20 (Tween 20) (0.05 mg/ml) Example I3: surfactant = polyethylene glycol (2) dodecyl ether (Brij L4) (0.05 mg/ml)

Example J

Insulin aspart 1000 U/ml Sodium phosphate 2 mM phenol 15.9 mM m-cresol 15.9 mM Ionic zinc (as ZnCl₂) 197 μg/ml (3 mM), equals 0.55% (w/w) based on the weight of insulin compound in the formulation TETA 5 mM Glycerol 174 mM Surfactant Selected from J1, J2 or J3 (see below) Water for injection qs Residual NaCl Acidification and subsequent neutralisation during preparation results in formation of 2-4 mM NaCl pH adjusted to 7.8 Example J1: surfactant = dodecyl maltoside (0.05 mg/ml) Example J2: surfactant = polysorbate 20 (Tween 20) (0.05 mg/ml) Example J3: surfactant = polyethylene glycol (2) dodecyl ether (Brij L4) (0.05 mg/ml)

Method for Preparation for the Above Formulations:

Insulin powder is added to water and HCl is added until the powder is fully dissolved (pH has to be <3 in order to achieve full dissolution). ZnCl2 is added to the required level. Once dissolved, pH is adjusted to approximately 7 and volume is adjusted with water so that the insulin concentration is 2× the required concentration. The composition is then mixed 1:1 (v/v) with a mixture of additional excipients (all at 2× the required concentration).

Example 2—Effect of Dodecyl Maltoside and Polysorbate 80 on the Stability of Insulin Aspart (1000 U/Ml) in the Presence of Trisodium Citrate, L-Histidine and Pyrophosphate

Stability of insulin aspart (1000 U/ml) was investigated in formulations comprising trisodium citrate (44 mM), L-histidine (22 mM) or pyrophosphate (22 mM), both in the presence and in the absence of dodecyl maltoside or polysorbate 80. All compositions (except control based on NovoRapid® composition, see below) further comprised phenol (15.9 mM), m-cresol (15.9 mM), sodium phosphate (2 mM), glycerol (174 mM), sodium chloride (10 mM) and ionic zinc (197 μg/ml, excluding counter-anion, as ZnCl2) and were adjusted to pH 7.4.

For comparison, a formulation of insulin aspart (1000 U/ml) in the composition of the 100 U/ml commercial insulin aspart product (NovoRapid®) was also included in the study. This formulation was prepared using the same procedure as that used for all other 1000 U/ml formulations studied in this experiment and contained the excipients of the commercial NovoRapid® product. The concentration of ionic zinc was adjusted to ensure the ratio between insulin aspart and ionic zinc was the same as that in the 100 U/ml NovoRapid® product. The formulation thus comprised sodium phosphate (7 mM), glycerol (174 mM), sodium chloride (10 mM), phenol (15.9 mM), m-cresol (15.9 mM) and ionic zinc (197 μg/ml, excluding counter-anion) and was adjusted to pH 7.4.

It was shown (Table 1) that the presence of trisodium citrate, L-histidine or pyrophosphate resulted in a considerable increase in the rate of particle formation of insulin aspart, using the Visual Assessment Scoring Method B. The presence of dodecyl maltoside mitigated the destabilising effect. Polysorbate 80 also showed a stabilising effect, although not to the same extent as dodecyl maltoside.

TABLE 1 Visual scores of insulin aspart (1000 U/ml) formulations using Visual Assessment Scoring Method B following storage at indicated temperatures. Ionic strength* T = 0 2-8° C. 30° C. 30° C. 37° C. Accelerator Surfactant (mM) weeks (12 weeks) (4 weeks) (12 weeks) (4 weeks) None None 24.16 1 1 2 2 3 Citrate None 24.16 1 2 4 5 5 (44 mM) Citrate Dodecyl maltoside 24.16 1 1 1 2 3 (44 mM) (50 μg/ml) Citrate Polysorbate 80 24.16 1 2 1 3 5 (44 mM) (50 μg/ml) Histidine None 24.16 1 2 4 5 5 (22 mM) Histidine Dodecyl maltoside 24.16 1 1 2 3 4 (22 mM) (50 μg/ml) Histidine Polysorbate 80 24.16 1 2 4 5 4 (22 mM) (50 μg/ml) Pyrophosphate None 24.16 1 3 5 5 5 (22 mM) Pyrophosphate Dodecyl maltoside 24.16 1 1 2 3 4 (22 mM) (50 μg/ml) Pyrophosphate Polysorbate 80 24.16 1 1 4 5 5 (22 mM) (50 μg/ml) NovoRapid ® control 35.83 1 1 2 2 3 (formulated at 1000 U/ml) *ionic strength calculation takes into account all ions in the formulation except for the zinc binding species (trisodium citrate, L-histidine or pyrophosphate) and the insulin compound using formula Ia.

Example 3—Effect of NaCl Concentration on the Stability of Insulin Aspart (1000 U/Ml) Both in the Presence and in the Absence of Trisodium Citrate/Dodecyl Maltoside Combination

The effect of NaCl concentration on the stability of insulin aspart (1000 U/ml) was investigated both in the presence and in the absence of trisodium citrate (44 mM)/dodecyl maltoside (50 μg/ml) combination. All formulations further comprised phenol (15.9 mM), m-cresol (15.9 mM), sodium phosphate (2 mM), ionic zinc (197 μg/ml, excluding counter-anion, as ZnCl2) and were adjusted to pH 7.4.

The formulations comprised either glycerol (174 mM) or NaCl (150 mM) or a mixture of glycerol and NaCl as a tonicity modifier (See Table 2). The concentration of glycerol in the formulations comprising a mixture of glycerol and NaCl was less than 174 mM so that the overall osmolarity of the compositions remained the same as in the compositions comprising glycerol only.

It was shown (Table 2) that the stability of insulin aspart (1000 U/ml) was negatively impacted by the presence of NaCl, both in the absence and in the presence of trisodium citrate (44 mM)/dodecyl maltoside (50 μg/ml) combination. In the absence of the trisodium citrate (44 mM)/dodecyl maltoside (50 μg/ml) combination, the stability was comparable using glycerol (174 mM) and glycerol (154 mM)/NaCl (10 mM) mixture as a tonicity modifier. However, considerable impairment in stability was observed when 150 mM NaCl was used. Interestingly, the impairment was observed only at 2-8° C. where a marked increase in the rate of particle formation was observed in the presence of 150 mM NaCl. The detrimental impact of increasing NaCl concentration on the stability of insulin aspart (1000 U/ml) was also observed in the presence of trisodium citrate (44 mM)/dodecyl maltoside (50 μg/ml) combination. Whilst only a small difference was observed between compositions comprising glycerol (174 mM) and glycerol (154 mM)/NaCl (10 mM) mixture as tonicity modifiers, a composition comprising glycerol (154 mM)/NaCl (50 mM) mixture showed a considerably impaired stability at 2-8° C.

It was thus demonstrated that increasing the ionic strength of the composition of insulin aspart at 1000 U/ml leads to an increased rate of particle formation.

Interestingly, this effect is not observed at lower concentrations (e.g. 100 U/ml) of insulin aspart, where an increase in the ionic strength of the composition can actually improve the stability of the formulation (data not shown). Similarly, for insulin lispro, while maintaining a low ionic strength at 1000 U/ml concentration provides improved stability, this effect is not observed a lower concentrations of insulin lispro (e.g. 100 U/ml) (data not shown).

TABLE 2 Visual scores of insulin aspart (1000 U/ml) formulations using Visual Assessment Scoring Method B following storage at indicated temperatures. Dodecyl Ionic maltoside strength* T = 0 2-8° C. 30° C. 30° C. 37° C. Citrate Tonicity modifier (μg/ml) (mM) weeks (12 weeks) (4 weeks) (12 weeks) (4 weeks) 0 mM Glycerol (174 mM) 0 14.16 1 1 1 2 3 0 mM Glycerol (154 mM) + 0 24.16 1 1 2 2 3 NaCl (10 mM) 0 mM NaCl (150 mM) 0 164.16 1 5 2 2 2 44 mM Glycerol (174 mM) 50 14.16 1 1 1 2 3 44 mM Glycerol (154 mM) + 50 24.16 1 1 1 2 3 NaCl (10 mM) 44 mM Glycerol (74 mM) + 50 64.16 1 5 3 3 5 NaCl (50 mM) *ionic strength calculation takes into account all ions in the formulation except for the zinc binding species (trisodium citrate) and the insulin compound using formula Ia.

Example 4: Comparison of the Source of Citrate and the pH of the Formulation on the Stability of Insulin Aspart (1000 U/Ml)

The effect of the source of citrate anion and the pH of the formulation on the stability of insulin aspart (1000 U/ml) was investigated. Citric acid and trisodium citrate were compared as the source of the citrate anion. The formulation comprising citric acid was tested at pH 7.8 and the formulation comprising trisodium citrate was tested at pH 7.4. Both formulations further comprised phenol (15.9 mM), m-cresol (15.9 mM), sodium phosphate (2 mM), glycerol (174 mM), dodecyl maltoside (50 μg/ml) and ionic zinc (197 μg/ml, excluding counter-anion, as ZnCl2).

It was shown (Table 3) that the source of citrate and the pH had a minimal impact on the stability of insulin aspart. The formulation comprising citric acid (pH 7.8) appeared to be very slightly more stable at the 8 weeks time-point at 30° C.

TABLE 3 Visual scores of insulin aspart (1000 U/ml) formulations using Visual Assessment Scoring Method B following storage at indicated temperatures. Ionic Source of strength* T = 0 2-8° C. 30° C. 30° C. 37° C. citrate anion pH (mM) weeks (8 weeks) (4 weeks) (8 weeks) (4 weeks) Citric acid 7.8 14.84 1 1 1 2 3 (44 mM) Trisodium citrate 7.4 14.16 1 1 1 3 3 (44 mM) *ionic strength calculation takes into account all ions in the formulation except for the zinc binding species (trisodium citrate, citric acid) and the insulin compound using formula Ia.

Example 5: Investigation of the Effect of Citric Acid Concentration on the Stability of Insulin Aspart (1000 U/Ml) in the Presence of Dodecyl Maltoside

The effect of citric acid concentration on the stability of insulin aspart (1000 U/ml) was investigated in the presence of dodecyl maltoside (0.05 mg/ml). All formulations tested further comprised phenol (15.9 mM), m-cresol (15.9 mM), sodium phosphate (2 mM), glycerol (174 mM), dodecyl maltoside (0.05 mg/ml) and ionic zinc (197 μg/ml, excluding counter-anion, as ZnCl2) and were adjusted to pH 7.8.

It was shown (Table 4) that increasing the concentration of citric acid from 0 to 44 mM had only a very small impact on the stability of insulin aspart (1000 U/ml) in the presence of dodecyl maltoside (0.05 mg/ml). No effect was observed at 2-8° C. and 37° C. for the duration of the experiment, and the rate of particle formation was only very slightly higher in the compositions comprising 22, 33 and 44 mM citric acid compared with compositions comprising 0 and 11 mM citric acid at 30° C.

TABLE 4 Visual scores of insulin aspart (1000 U/ml) formulations using Visual Assessment Scoring Method B following storage at indicated temperatures. Ionic Citric strength* T = 0 2-8° C. 30° C. 30° C. 37° C. acid (mM) weeks (8 weeks) (4 weeks) (8 weeks) (4 weeks)  0 mM 14.84 1 1 1 1 3 11 mM 14.84 1 1 1 1 3 22 mM 14.84 1 1 1 2 3 33 mM 14.84 1 1 1 2 3 44 mM 14.84 1 1 1 2 3 *ionic strength calculation takes into account all ions in the formulation except for the zinc binding species (citric acid) and the insulin compound using formula Ia.

Example 6: Investigation of the Optimal Concentration of Dodecyl Maltoside and Polysorbate 80 on the Stability of Insulin Aspart (1000 U/Ml) in the Presence of Different Concentrations of Citric Acid

The stability of insulin aspart (1000 U/ml) was investigated in the presence of different concentrations of citric acid and different concentrations of either dodecyl maltoside or polysorbate 80. All formulations tested further comprised phenol (15.9 mM), m-cresol (15.9 mM), sodium phosphate (2 mM), glycerol (174 mM) and ionic zinc (197 μg/ml, excluding counter-anion, as ZnCl2) and were adjusted to pH 7.8. Three concentrations of citric acid (44, 66 and 88 mM) and four concentrations of each non-ionic surfactant were tested as well as corresponding surfactant-free compositions.

The rate of particle formation in formulations of insulin aspart (1000 U/ml) was found to be proportional to citric acid concentration in the range between 44 and 88 mM, with the lower citric acid concentration of 44 mM being most suitable (Table 5). Whilst the presence of both dodecyl maltoside and polysorbate 80 led to a reduction in the rate of particle formation, dodecyl maltoside was found more effective in inhibiting the particle formation than polysorbate 80. The lower concentrations of dodecyl maltoside (0.05 and 0.1 mg/ml) appeared to be more effective in inhibiting the particle formation than higher concentrations (0.2 and 0.3 mg/ml). In contrast, in the case of polysorbate 80 it was the higher concentrations (0.3 and 0.5 mg/ml) that showed a greater ability to reduce the particle formation rate than the lower concentrations (0.05 and 0.1 mg/ml).

TABLE 5 Visual scores of insulin aspart (1000 U/ml) formulations using Visual Assessment Scoring Method B following storage at indicated temperatures. Dodecyl Polysorbate Ionic Citric maltoside 80 strength * T = 0 2-8° C. 30° C. 30° C. 37° C. acid (mg/ml) (mg/ml) (mM) weeks (8 weeks) (4 weeks) (8 weeks) (4 weeks) 44 mM 0 0 14.84 1 3 4 5 5 44 mM 0.05 0 14.84 1 1 1 2 3 44 mM 0.1 0 14.84 1 1 1 2 3 44 mM 0.2 0 14.84 1 1 2 2 4 44 mM 0.3 0 14.84 1 2 2 3 5 44 mM 0 0.05 14.84 1 3 2 3 4 44 mM 0 0.1 14.84 1 2 2 3 4 44 mM 0 0.3 14.84 1 2 2 3 4 44 mM 0 0.5 14.84 1 1 1 3 4 66 mM 0 0 14.84 1 5 5 5 5 66 mM 0.05 0 14.84 1 2 2 4 4 66 mM 0.1 0 14.84 1 3 2 3 4 66 mM 0.2 0 14.84 1 3 2 5 5 66 mM 0.3 0 14.84 1 4 3 5 5 66 mM 0 0.05 14.84 1 5 4 5 5 66 mM 0 0.1 14.84 1 5 4 5 5 66 mM 0 0.3 14.84 1 4 3 4 4 66 mM 0 0.5 14.84 1 4 4 5 5 88 mM 0 0 14.84 1 5 5 5 5 88 mM 0.05 0 14.84 1 4 2 4 5 88 mM 0.1 0 14.84 1 5 3 3 5 88 mM 0.2 0 14.84 1 5 4 5 5 88 mM 0.3 0 14.84 1 5 4 5 5 88 mM 0 0.05 14.84 1 5 4 5 5 88 mM 0 0.1 14.84 1 5 4 4 5 88 mM 0 0.3 14.84 1 5 3 4 5 88 mM 0 0.5 14.84 1 5 3 5 5 * ionic strength calculation takes into account all ions in the formulation except for the zinc binding species (citric acid) and the insulin compound using formula Ia.

Example 7—Comparison of Pharmacodynamic and Pharmacodynamic Profiles of Insulin Aspart (100 and 1000 U/Ml) Formulations in the Presence and in the Absence of Citrate and Dodecyl Maltoside

Pharmacodynamic and pharmacokinetic profile of insulin aspart was compared in the following compositions using the Diabetic Pig Pharmacokinetic/Pharmacodynamic Model (see General Methods (c)):

-   -   Insulin aspart (100 U/ml) in the formulation of the currently         marketed NovoRapid® (100 U/ml) rapid-acting product     -   Insulin aspart (1000 U/ml) in the formulation of the currently         marketed NovoRapid® (100 U/ml) rapid-acting product     -   Insulin aspart (1000 U/ml) in a formulation of the invention         comprising 22 mM trisodium citrate and 0.1 mg/ml dodecyl         maltoside     -   Insulin aspart (1000 U/ml) in a formulation of the invention         comprising 44 mM trisodium citrate and 0.1 mg/ml dodecyl         maltoside

All formulations tested comprised phenol (15.9 mM) and m-cresol (15.9 mM) and were adjusted to pH 7.4. The additional components of each formulation are listed in Table 6.

TABLE 6 Additional components in formulations of insulin aspart tested. Insulin Sodium Ionic Trisodium Dodecyl aspart phosphate NaCl Glycerol zinc* citrate maltoside Formulation (U/ml) (mM) (mM) (mM) (μg/ml) (mM) (mg/ml) 7A 100 7 10 174 19.7 7B 1000 7 10 174 197 7C 1000 2 150 197 22 0.1 7D 1000 2 150 197 44 0.1 *Does not include the contribution of counter-anion

Pharmacodynamic profiles of formulations 7A-7D are shown in FIG. 1. It was shown that increasing the concentration of insulin aspart from 100 U/ml to 1000 U/ml in the formulation of the marketed NovoRapid® product led to a slower onset of action. This is in line with previous reports of dose-dependent delays of the glucose reduction effect of rapid-acting insulins (e.g. de la Pefia et al. Pharmacokinetics and Pharmacodynamics of High-Dose Human Regular U-500 Insulin Versus Human Regular U-100 Insulin in Healthy Obese Subjects, Diabetes Care, 34, pp 2496-2501, 2011). It was also shown (FIG. 1) that a formulation of insulin aspart (1000 U/ml) comprising 44 mM trisodium citrate and 0.1 mg/ml dodecyl maltoside resulted in a pharmacodynamic profile that was comparable with that achieved by the formulation of the marketed NovoRapid® product (100 U/ml). Such acceleration of the onset of the glucose reduction was not observed in a composition comprising 22 mM trisodium citrate and 0.1 mg/ml dodecyl maltoside, indicating that this concentration of citrate is too low to achieve the accelerating effect at this concentration of insulin aspart.

The pharmacokinetic profiles of formulations 7A, 7B and 7D (FIG. 2) were in line with the pharmacodynamic profiles, showing that increasing the concentration of insulin aspart from 100 U/ml to 1000 U/ml in the formulation of the marketed NovoRapid® product led to a slower increase in serum insulin level, whereas the formulation comprising 44 mM trisodium citrate and 0.1 mg/ml dodecyl maltoside resulted in a profile that was comparable with that achieved by the formulation of the marketed NovoRapid® product (100 U/ml). The pharmacokinetic profile of Formulation 7C was not tested.

The T_(MAX) and T_(1/2MAX) mean values and standard deviations (SD) relating to the pharmacokinetic profiles of formulations 7A, 7B and 7D are shown in Table 7 below.

TABLE 7 T_(MAX) and T_(1/2MAX) mean values and standard deviations (SD) relating to the pharmacokinetic profiles of formulations 7A, 7B and 7D. T_(MAX) (mean) T_(MAX) (SD) T_(1/2MAX) (mean) T_(1/2MAX) (SD) 7A 25.71 8.38 8.01 2.35 7B 90.83 21.68 28.67 8.02 7D 20.71 6.07 7.00 3.53

Results of the Student's t-test performed to evaluate bioequivalence between formulations 7A, 7B and 7D are shown in Table 8 below. Formulation 7A and 7D were shown to be bioequivalent, whereas formulations 7A and 7B and formulations 7B and 7D were shown to be non-bioequivalent.

TABLE 8 Bioequivalence t-test analysis of the pharmacokinetic profiles of formulations 7A, 7B and 7D. T_(MAX) p-value T_(1/2MAX) p-value 7A vs 7B 0.0118 0.0115 7A vs 7D 0.2507 0.3762 7B vs 7D 0.0177 0.0107

Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

All publications, patents, patent applications, internet sites, and accession numbers/database sequences (including both polynucleotide and polypeptide sequences) cited are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference.

SEQUENCE LISTING SEQ ID NO: 1: GIVEQCCTSICSLYQLENYCN SEQ ID NO: 2: FVNQHLCGSHLVEALYLVCGERGFFYTPKT SEQ ID NO: 3: FVNQHLCGSHLVEALYLVCGERGFFYTKPT SEQ ID NO: 4: FVNQHLCGSHLVEALYLVCGERGFFYTDKT SEQ ID NO: 5: FVKQHLCGSHLVEALYLVCGERGFFYTPET 

1. An aqueous liquid pharmaceutical formulation comprising (i) an insulin compound at a concentration of 500-1000 U/ml, (ii) ionic zinc, (iii) a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C., and (iv) a non-ionic surfactant; and wherein the formulation is substantially free of EDTA and any other zinc binding species having a logK with respect to zinc ion binding of more than 12.3 at 25° C.
 2. The formulation according to claim 1, wherein the ionic strength of the formulation is less than 40 mM, said ionic strength being calculated using the formula: $\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$ in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x) is the absolute value of the charge of ion x and the sum covers all ions (n) present in the formulation except for the zinc binding species and the insulin compound.
 3. The formulation according to claim 2, wherein the ionic strength of the formulation using formula Ia is less than 30 mM.
 4. The formulation according to claim 1, wherein the non-ionic surfactant is other than polysorbate
 80. 5. The formulation according to claim 1, wherein the insulin compound is not insulin glargine.
 6. The formulation according to claim 1, wherein the insulin compound is insulin lispro, insulin glulisine, or recombinant human insulin.
 7. The formulation according to claim 1, wherein the insulin compound is insulin aspart.
 8. (canceled)
 9. (canceled)
 10. The formulation according to claim 1, wherein the insulin compound is present at a concentration of >500-1000 U/ml.
 11. The formulation according to claim 10, wherein the insulin compound is present at a concentration of 1000 U/ml.
 12. The formulation according to claim 1, wherein the ionic zinc is present at a concentration of: more than 0.05%, more than 0.5%, or 0.5-1% by, weight of zinc based on the weight of insulin compound in the formulation.
 13. (canceled)
 14. (canceled)
 15. The formulation according to claim 1, wherein the zinc binding species having a logK with respect to zinc ion binding in the range 4.5-12.3 is selected from citrate, pyrophosphate, aspartate, glutamate, cysteine, cystine, glutathione, ethylenediamine, histidine, DETA and TETA.
 16. The formulation according to claim 15, wherein the zinc binding species is citrate.
 17. The formulation according to claim 16, wherein the source of the citrate is citric acid.
 18. The formulation according to claim 1, wherein the zinc binding species having a logK with respect to zinc ion binding in the range 4.5-12.3 is present at a concentration of 1-50 mM.
 19. The formulation according to claim 18, wherein the zinc binding species having a logK with respect to zinc ion binding in the range 4.5-12.3 is present at a concentration of 10-50 mM.
 20. The formulation according to claim 1, wherein the molar ratio of ionic zinc to zinc binding species is 1:3 to 1:175.
 21. The formulation according to claim 1, wherein the zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C. is a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-10 at 25° C.
 22. The formulation according to claim 1, which is substantially free of zinc binding species having a logK with respect to zinc ion binding of 10-12.3 at 25° C.
 23. The formulation according to claim 1, wherein the non-ionic surfactant is an alkyl glycoside.
 24. The formulation according to claim 23, wherein the alkyl glycoside is selected from the group consisting of dodecyl maltoside, dodecyl glucoside, octyl glucoside, octyl maltoside, decyl glucoside, decyl maltoside, decyl glucopyranoside, tridecyl glucoside, tridecyl maltoside, tetradecyl glucoside, tetradecyl maltoside, hexadecyl glucoside, hexadecyl maltoside, sucrose monooctanoate, sucrose monodecanoate, sucrose monododecanoate, sucrose monotridecanoate, sucrose monotetradecanoate and sucrose monohexadecanoate.
 25. The formulation according to claim 24, wherein the alkyl glycoside is dodecyl maltoside or decyl glucopyranoside.
 26. The formulation according to claim 25, wherein the alkyl glycoside is dodecyl maltoside.
 27. The formulation according to claim 1, wherein the non-ionic surfactant is a polysorbate surfactant.
 28. The formulation according to claim 1, wherein the non-ionic surfactant is an alkyl ether of polyethylene glycol.
 29. The formulation according to claim 28, wherein the alkyl ether of polyethylene glycol is selected from polyethylene glycol (2) dodecyl ether, polyethylene glycol (2) oleyl ether and polyethylene glycol (2) hexadecyl ether.
 30. The formulation according to claim 1, wherein the non-ionic surfactant is a block copolymer of polyethylene glycol and polypropylene glycol.
 31. The formulation according to claim 30, wherein the block copolymer of polyethylene glycol and polypropylene glycol is poloxamer 188, poloxamer 407, poloxamer 171 or poloxamer
 185. 32. The formulation according to claim 1, wherein the non-ionic surfactant is an alkylphenyl ether of polyethylene glycol.
 33. The formulation according to claim 32, wherein the alkylphenyl ether of polyethylene glycol is 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol.
 34. The formulation according to claim 1, wherein the non-ionic surfactant is present at a concentration of 1-1000 μg/ml.
 35. The formulation according to claim 34, wherein the non-ionic surfactant is present at a concentration of 10-400 μg/ml.
 36. The formulation according to claim 1, further comprising an uncharged tonicity modifying agent selected from the group: trehalose, mannitol, glycerol and 1,2-propanediol.
 37. (canceled)
 38. The formulation according to claim 36, wherein the uncharged tonicity modifying agent is glycerol.
 39. The formulation according to claim 1, wherein the formulation comprises <10 mM chloride.
 40. The formulation according to claim 1, wherein the composition is substantially isotonic.
 41. The formulation according to claim 1, wherein the pH is in the range 5.5 to 9.0.
 42. The formulation according to claim 41, wherein the pH is in the range 7.0 to 7.5 or 7.6 to 8.0.
 43. (canceled)
 44. A formulation according to claim 42, which comprises a phosphate buffer.
 45. The formulation according to claim 1, further comprising a preservative selected from the group: phenol, m-cresol, chlorocresol, benzyl alcohol, propylparaben, methylparaben, benzalkonium chloride and benzethonium chloride.
 46. (canceled)
 47. A formulation according to claim 1, further comprising at least one of nicotinamide, nicotinic acid or a salt thereof, and treprostinil or a salt thereof.
 48. (canceled)
 49. (canceled)
 50. An aqueous liquid pharmaceutical formulation according to claim 1, comprising (i) an insulin compound at a concentration of 500-1000 U/ml (ii) ionic zinc, (iii) citrate as a zinc binding species at a concentration of 1 mM or more, and (iv) a non-ionic surfactant; wherein the formulation is substantially free of EDTA and any other zinc binding species having a logK with respect to zinc ion binding of more than 12.3 at 25° C., and wherein the ionic strength of the formulation is less than 40 mM, said ionic strength being calculated using the formula: $\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$ in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x) is the absolute value of the charge of ion x and the sum covers all ions (n) present in the formulation except for the zinc binding species and the insulin compound.
 51. A formulation according to claim 50, wherein the citrate is present in the formulation at a concentration of 30-50 mM.
 52. (canceled)
 53. A method of treatment of diabetes mellitus which comprises administering to a subject in need thereof an effective amount of a formulation according to claim
 1. 54. A container containing one dose or a plurality of doses of the formulation according to claim
 1. 55. An injection device for single or multiple use comprising a container containing one dose or a plurality of doses of the formulation according to claim 1, together with an injection needle.
 56. A medical device comprising a reservoir comprising a plurality of doses of the formulation according to claim 1 and a pump adapted for automatic or remote operation such that upon automatic or remote operation one or more doses of the formulation is administered to the body.
 57. A dry solid pharmaceutical composition suitable for reconstitution with an aqueous medium which comprises, following reconstitution, (i) an insulin compound at a concentration of 500-1000 U/ml, (ii) ionic zinc, (iii) a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C., and (iv) a non-ionic surfactant; and wherein the composition is substantially free of EDTA and any other zinc binding species having a logK with respect to zinc ion binding of more than 12.3 at 25° C.
 58. A method of preparing a formulation according to claim 1 which comprises dissolving a dry solid pharmaceutical composition according to claim 57 in an aqueous medium.
 59. A method of improving the storage stability of an aqueous liquid pharmaceutical formulation comprising (i) an insulin compound at a concentration of 500-1000 U/ml, (ii) ionic zinc and (iii) a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C.; wherein the formulation is substantially free of EDTA and any other zinc binding species having a logK with respect to zinc ion binding of more than 12.3 at 25° C.; which comprises adding a non-ionic surfactant to the formulation.
 60. (canceled)
 61. A method of accelerating the onset of action of an aqueous liquid pharmaceutical formulation comprising (i) an insulin compound at a concentration of 500-1000 U/ml, (ii) ionic zinc, and (iii) a non-ionic surfactant; which comprises adding to the formulation a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C.
 62. (canceled)
 63. The composition of claim 57, wherein the ionic strength of the formulation is less than 40 mM, said ionic strength being calculated using the formula: $\begin{matrix} {I = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}} & {Ia} \end{matrix}$ in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x) is the absolute value of the charge of ion x and the sum covers all ions (n) present in the formulation except for the zinc binding species and the insulin compound.
 64. The composition of claim 63, wherein the ionic strength calculated using formula Ia is less than 30 mM.
 65. A composition according to claim 57, wherein the non-ionic surfactant is other than polysorbate 80; and/or wherein the zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-12.3 at 25° C. is a zinc binding species at a concentration of 1 mM or more selected from species having a logK with respect to zinc ion binding in the range 4.5-10 at 25° C.; and/or wherein the formulation is substantially free of zinc binding species having a logK with respect to zinc ion binding of 10-12.3 at 25° C.
 66. (canceled)
 67. (canceled)
 68. A composition according to claim 57, wherein the zinc binding species is citrate.
 69. A formulation according to claim 1, wherein the formulation does not contain a vasodilator, nicotinamide, nicotinic acid or a salt thereof.
 70. A formulation according to claim 1, wherein the formulation further comprises an additional therapeutically active agent of use in the treatment of diabetes.
 71. A formulation according to claim 70, wherein the additional agent is an amylin analogue or a GLP-1 agonist.
 72. A formulation according to claim 1, wherein the formulation is co-administered with a long acting insulin. 